Reigning Madrid Open champion Iga Swiatek suffered a crushing semi-final defeat by Coco Gauff on Thursday, with the American triumphing 6-1, 6-1.
The fourth-seeded Djokovic suffered a second consecutive opening defeat, after losing his opener against Alejandro Tabilo in Monte Carlo two weeks ago.
World number three Carlos Alcaraz withdrew from the Madrid Open on Thursday with a thigh injury, in a blow for his French Open title defence preparations.
Rublev, who takes the Madrid crown from double champion Carlos Alcaraz whom he beat in the quarter-finals, said he had played despite feeling ill at times this week and hailed his doctors for helping him through.
Coming back from 1-3 down in the decider and saving three match points in total, Swiatek claimed a 7-5, 4-6, 7-6 (9/7) victory to secure the Madrid Open trophy for the first time.
Rublev upset home favourite Alcaraz 4-6, 6-3, 6-2 to reach the semi-finals.
On the women's side, Iga Swiatek was made to sweat in her 4-6, 6-0, 6-2 victory over Brazilian lefty Beatriz Haddad Maia, as she dropped her first set of the tournament and needed two and a half hours to reach a second consecutive Madrid semi-final.
Jabeur, the 2022 Madrid champion, took a swipe at tournament organisers, telling reporters she believes tennis still has a way to go to achieve gender equality, despite it being the leader in women's sport.
Defending Madrid Open champions Carlos Alcaraz and Aryna Sabalenka both triumphed in their opening clashes in the Spanish capital on Friday to reach the third round.
Djokovic has now pulled out of the Madrid Open three times in four years.
The Spaniard earned his 10th career title with a hard-fought 6-4, 3-6, 6-3 win over Jan-Lennard Struff and can reclaim the world number one ranking from Novak Djokovic simply by playing a single match at the Rome Masters next week, before the French Open.
The big-hitting Belarusian gained revenge for defeat by her Polish rival in the Stuttgart final a fortnight ago with a thrilling triumph to end Swiatek's nine-match winning streak ahead of her French Open defence
Defending champion Ons Jabeur said on Monday she had withdrawn from the Madrid Open with a calf injury after retiring from the Stuttgart Open at the weekend.
Both men are vying for a record-extending 23rd Grand Slam title at Roland Garros, with the tournament beginning on May 28
Eugenie Bouchard pulled off her first win over Maria Sharapova with a 7-5 2-6 6-4 triumph in the Madrid Open on Monday in a thrilling contest full of tension after the Canadian had called the Russian a cheater following her doping ban.
