Formed in January 1800 as the "Experimental Corps of Riflemen" to provide sharpshooters, scouts and skirmishers, they were soon renamed the "Rifle Corps". In January 1803 they became an established regular regiment and were titled the 95th Regiment of Foot (Rifles). The unit was distinguished by its use of green uniforms as standard in place of the traditional redcoat, as well as being armed with the first British-made rifle accepted by the British Army, in place of smooth-bore muskets — the first regular infantry corps in the British Army to be so.
In August 1808 the 2nd/95th was part of the immediate forces sent in the Portuguese expedition initially commanded by Sir Arthur Wellesley and covered the landings at Montego Bay. On 15 August they had the distinction of firing the first shots of the Peninsular War during a skirmish at Óbidos against the French, but also unfortunately suffered the first British officer fatality of the war, a Lieutenant Ralph Bunbury. The 95th, as part of 6th Brigade which included the rifle armed 5th/60th Foot, took part in the Battle of Roliça, the first pitched battle of the war, on 17 August 1808. Rifleman Thomas Plunket of the 1st Battalion, 95th Rifles, shot the French General Auguste François-Marie de Colbert-Chabanais at a range of up to 800 yards (730 m) at the Battle of Cacabelos on 3 January 1809.
The 1st battalion was part of John Moore's campaign which ended with evacuation after the Battle of Corunna on 16 January 1809. The majority of the 1st battalion was rested and refitted in the UK, though a few small detachments of the 95th were stranded behind which then formed up with other detachments as part of a defence force (1st Battalion of Detachments) in Portugal. The 1st returned to the peninsula a few months later in May 1809 and in July was force marched in an attempt to arrive with the main force for the Battle of Talavera but despite covering a notable distance they arrived on the 29 July 1809, just after the battle.
After the depletion suffered at Corunna, the two battalions of the 95th based at Hythe in Kent were made up to a strength of 1,000 men each. However, so many volunteers came forward to join the regiment that permission was granted to raise a third battalion in 1809
The regiment had already become so famous and popular, that not only were the deficiencies filled up in a very short time, but more than a thousand volunteers presented themselves beyond the numbers required. It was therefore resolved by the Authorities to add a 3rd Battalion to the regiment.
The third battalion joined the Peninsular Army in 1810. Thereafter the three battalions of the 95th fought in numerous major battles and skirmishes during the Peninsular War as part of the elite Light Division, including the Battle of Bussaco in September 1810 and the sieges of Ciudad Rodrigo in January 1812 and Badajoz and March 1812 as well as the Battle of Vitoria in June 1813. At the Battle of San Marcial in August 1813 a company of the 95th Rifles under the command of Captain Daniel Cadoux held off an entire French division at Vera before withdrawing. The regiment also took part in the Battle of Nivelle in November 1813.
The three battalions had been dispersed to various locations with the abdication of Napoleon and the total French defeat in 1814. The majority of the regiment's companies were sent back to England for rest and refitting while several companies had been retained in north-east France at Leuze, Aisne under General Thomas Graham. Five companies of the 3rd battalion were in North America, having been sent in late 1814 to participate in the final stages of the War of 1812. With the return of Napoleon from exile, all of the companies in England crossed the channel and landed in Belgium in May 1815, joining with those already present, so that the entire regiment, bar the five companies still in America, became part of Wellington's Anglo-Dutch army. The first battalion went on to fight at the Battle of Quatre Bras on 16 June 1815, while all three battalions would fight at the Battle of Waterloo on 18 June 1815.
At the end of the Napoleonic Wars, the size of the British Army was reduced; in line with precedent the more recently formed regiments were disbanded first. The unique skills of the 95th were considered too valuable to lose so the 95th, having seen distinguished service in the Napoleonic Wars, was taken out of line of the British Army and became the "Rifle Brigade" on 23 February 1816 (the number was reassigned eight years later to the newly formed county regiment of the 95th (Derbyshire) Regiment of Foot). The Duke of Wellington became Colonel-in-Chief of the regiment in 1820 and served until his death.