In a contest that had nothing but pride on the line, both sides showed plenty of grit and attacking intent, with the game producing seven tries and going right down to the wire.
The hosts struck first through a brilliant piece of interplay, with Edwill van der Merwe finishing a flowing move to put the Lions 5-0 ahead after an earlier effort from Lubabalo Dobela was ruled out for a knock-on.
The Ospreys hit back almost immediately, capitalising on a charged-down exit kick to score a scrappy try through Will Griffiths. Dan Edwards converted to edge the visitors 7-5 ahead.
Despite dominating possession and territory, the Lions struggled to break down a resolute Ospreys defence. But eventually, captain Jaco Visagie powered over for the Lions' second try after sustained pressure. Quan Horn added the extras to give the hosts a 12-7 lead at the break.
The Ospreys came out with renewed energy, and after several phases of forward carries, James Ratti crashed over for a converted try to regain the lead at 14-2.
The Lions responded with a second for Visagie, who dotted down following a vital turnover from Renzo du Plessis and some slick lineout play. The conversion was missed, but the Lions had nudged ahead 17-14.
Back came the Ospreys, this time through fullback Jack Walsh, who cut a beautiful line to score under the posts, handing the visitors a 21-17 lead after Edwards' conversion.
The lead didn't last long. Henco van Wyk struck next with a well-worked move off a switch to hand the Lions a 24-21 advantage with Dobela converting.
With just five minutes left, the Ospreys won a kickable penalty, bravely opting to kick for touch. The gamble paid off as replacement hooker Sam Parry muscled his way over for a try, and Edwards added the extras to make it 28-24 in favour of the Welsh side.
But in the final minute, with the clock in the red and the Ellis Park crowd on their feet, the Lions earned a penalty and kicked to the corner. From the lineout, they launched one last assault, and it was Dobela who crashed over to steal the win in dramatic fashion. Though his conversion missed, it mattered not as Lions sealed a 29-28 victory.