Defending Varsity Football champions UP-Tuks booked a semifinal place with a 4-0 win over NMMU in Pretoria.
The defeat means the end of the season for the Madibaz who needed a draw to qualify for the play-offs.
UP-Tuks will travel to Mafikeng to face the unbeaten NWU in their semifinal next week, whilst UJ will host Kovsies in the other play-off match.
It was competitive from the word go as both sides moved the ball forward at pace in search of an early goal.
It would be the hosts to take control with a double strike as first Jeremiah Nkwana curled a free kick into the top right corner before Simbongile Njokwe buried a header from a corner to put them 2-0 up.
That left NMMU with it all to do and they started to enjoy more possession as they looked to fight their way back into the game.
However, the home side would extend their lead to 3-0 at half-time thanks to an own goal from NMMU defender Lundi Balfour who was trying to hold off a charging Richard Moremi.
The visitors threw everything at their hosts in the second half with their play-off hopes slipping through their hands, but could not break down the stubborn UP-Tuks defence.
Opportunities were few and far between after half-time and UP-Tuks had the final say as Thabo Ndlovu came up with a goal in extra time.
FNB man of the match: Richard Moremi
Samsung Super Striker: Jeremiah Nkwana
For UP-Tuks: Jeremiah Nkwana, Simbongile Njokwe, Lundi Balfour (og), Thabo Ndlovu
UP-Tuks: 32 Lebo Ngubeni, 4 Lawrence Ntswane, 8 Nicholas Killas, 13 Lindsay Stowman, 20 Thabo Ndlovu, 10 Jeremiah Nkwana, 11 Richard Moremi, 5 Simbongile Njokwe, 15 Jarryd Van Den Berg, 9 Mbongeni Masilela, 18 Lucky Mathosi.
Substitutes: 1 Dylan Paterson, 6 Khumbulani Siluma, 12 Phuthego Mabitsela, 7 Luke Steyl, 22 Dean Wilkinson, 2 Botshelo Mfulwane, 23 Vuze-Umuze Khoza.
NMMU: 24 Lyndsay Jennings, 12 Onela Beqezi, 5 Athini Bisha, 16 Lundi Balfour, 13 Vuyolwethu Marele, 19 Karabo Motlhabi, 6 Kurt Duff (captain), 10 Cloudius Sangandira, 18 Olwethu Ngako, 14 Simamkele Thobi, 8 Lukhanyo Rasmeni.
Substitutes: 1 Matthew Booysen, 21 Kevin Kevido, 4 Zukisani Mtendeni, 20 Litha Fingo, 7 Sachin Jackson, 11 Kieran Koert, 9 Lukhanyo Ndabeta.