Today's lung-busting trek to La Planche des Belles Filles has been labelled a duel between Italy's Nibali and Spaniard Alberto Contador since defending champion Chris Froome pulled out injured last week.
Nibali's coup de force on the tricky cobbles on the fifth stage mean he holds a two minute 34 second advantage over Contador with the big mountains looming.
Although Nibali lost three seconds to Contador in the first hilltop finish on Saturday as his team showed a first glimpse of weakness, Astana got rid of the burden of controlling the race yesterday when deciding not to chase a counter-attack group.
France's Tony Gallopin, who has no realistic hope of the overall title, took over the yellow jersey on stage nine and holds a 1:34 advantage over Nibali.
"We saw that Gallopin was in that group but we were not going to kill our team to keep the jersey," Astana team manager Alexandre Vinokourov said.
Astana now have no responsibility to control the pace of the peloton today while Nibali will be rested for the mountains.
"The most important thing was to save energy for tomorrow's stage. Tomorrow will be another great finale between Vincenzo and Alberto at La Planche des Belles Filles," Vinokourov said.
His sports director Giuseppe Martinelli was all smiles.
"Before the Tour we would have signed to find ourselves in that situation, to have 2:30 on Contador at that point of the race," he said.
Nibali is looking to become only the sixth man to win all three grand Tours (France, Spain, Italy) after Frenchmen Jacques Anquetil and Bernard Hinault, compatriot Felice Gimondi, Belgium's Eddy Merckx and Contador. He won the Vuelta in 2010 and the Giro in 2013.
Germany's Tony Martin powered to an impressive victory in yesterday's stage after a 155-km mountain raid in the Vosges.
Time-trial world champion Martin attacked after 15 km and dropped his breakaway companion with just under 60 left to win his third Tour stage following time trial victories in 2011 and 2013.
"It was perfect, there were no mind games in that breakaway," Martin said. "I just had to go, I knew I could ride very fast. Everything worked perfectly, I had really, really good legs, there were good conditions."
Gallopin, of the Lotto-Belisol team, now leads Nibali but has little hope of retaining that position.
"It's unbelievable. But it won't be the easiest stage for defending the lead," said Gallopin.
"Favourites will undergo their first real big test and I only have a one and half minute lead. I'll do all I can to keep the jersey. I'd love to have it for more than one day."
Several groups attacked early on, with Martin of the Omega Pharma-Quick Step team and Italian Alessandro De Marchi leading the way.
They were followed by a group of 28 featuring Pierre Rolland and Gallopin, who had been 3:27 behind Nibali at the start.
Martin's mammoth work helped the duo build a 6:30 lead over the peloton while the Gallopin group could not close the gap, gradually falling back despite the work of Rolland's Europcar team mates.
Martin powered away with 59 km left at the foot the first-category climb of the Col de Markstein, keeping the Gallopin group at bay while De Marchi struggled.
Swiss Fabian Cancellara took second place and Belgian Greg van Avermaet finished third in the stage.
Spain's Joaquim Rodriguez, third overall last year, who said before the race he was looking for stage wins only, signalled his intentions to claim the polka dot jersey for the mountain classification.
The Katusha rider, who was in the Gallopin group, grabbed points at top of the Markstein and Grand Ballon climbs.
Gallopin jumped away from the counter-attacking group in the descent of the Grand Ballon but was reined in with 10km left.