He confessed he was "nervous" before the simple finger-prick procedure at a London sexual health clinic.
It was the first time a member of the royal family had been publicly tested for HIV and was akin to the efforts of his late mother Diana, Princess of Wales, to break down the stigma surrounding the disease.
In the late 1980s, when many still believed the disease could be contracted through casual contact, she sat on the sickbed of a man with Aids and held his hand.
Ian Green, chief executive of the Terrence Higgins Trust, praised the prince for his "genuine and personal commitment to tackling HIV" and called it a "groundbreaking moment in the fight against HIV" which would help normalise testing and inspire a new generation to take control of their sexual health.
The test was performed by Robert Palmer, a lead health adviser and psychosexual counsellor, in a small cubicle at the Burrell Street centre near the Tate Modern museum.
It covers the boroughs of Lambeth and Southwark which have some of the highest rates of sexually transmitted infections - including HIV - in England, according to Public Health England.
Harry said before the procedure started: "Even though I'm not from this part of London, being the person that I am and the people that I end up being around - I'm still nervous, which is really interesting."
Mr Palmer replied: "It's not surprising because you're going to be doing a test and you're going to find out something about yourself, by having the test done."
Harry has recently made highlighting the issue of HIV/Aids a major element of his public work and had to wait a few moments while the sample was mixed with chemicals and poured into a test tray where a developing solution was added.
The negative result was indicated by a blue spot seen in the tray - if two spots had appeared he would have needed further tests to confirm if he was HIV positive.