The game ended at 3-3 after regulation time.
Matsatsantsa scored twice in the final 17 minutes to force extra-time, but following a goalless half-hour, SuperSport were the better side from 12 yards with Ronwen Williams making two penalty saves.
Leading up to the clash, a lot was made about AmaTuks poor record against their Tshwane rivals: after 12 minutes they scored their first competitive goal in what was their 10th game against SuperSport.
Having won back the ball inside their own area, AmaTuks launched a swift counter-attack and after Thabiso Nkoana hit the upright, Innocent Nemukondeni pounced to stroke home the rebound.
Micheal Morton brought the visitors back down to earth in the 26th minute – the defensive midfielder with a rare goal following a mistake by Washington Arubi.
Lennox Bacela will be integral to AmaTuks bid for survival this season, and having played a role in the opening goal, the former Orlando Pirates set-up the second. Ian Chikohwa restoring Tuks lead on 53 minutes.
AmaTuks extended their lead soon after in rather fortunate circumstances. Grant Kekana heading the goal into the back of his own net.
The visitors were on course for their first win against their Tshwane rivals, but SuperSport rallied and after Jeremy Brockie equalised with a near post header in the 73rd minute, Morne Nel prodded home the equaliser in the 84th minute.
Following a rather uneventful 30-minute period of extra-time, SuperSport won it from the spot with Brockie netting the winner.
The draw of the last eight will be made on Thursday.
