Arnaud Demare (FDJ) won the sprint finish to claim stage honours ahead of compatriots Yohann Gene (Direct Energie) and Stephane Poulhies (Equipe Cycliste Armée de Terre) following the 154.8km route from Gers to Astarac Arros en Gascogne.
The Route de Sud was Quintana's only Tour de France build-up race after he opted to skip the traditional week-long stage races and avoid key rivals at the Criterium du Dauphine and Tour de Suisse.
Quintana, who won the race for the second time, took control of the race by winning the hilly time-trial on Friday and consolidating that into a 36 second overall victory ahead of Movistar team-mate Marc Soler, who won the fourth stage, with Nicolas Edet (Cofidis) third.
“It’s a very important victory. I was coming into this event following long weeks of training in atlitude at home in Colombia, and I could confirm I’m in good form," Quintana said.
"Legs have gone as usual for me in the mountains and it seems obvious that I’ve improved quite a bit into the time trial.
“The team did really well: great job from them from the beginning to the end of the race, crowning it today after a mountain stage where they rode fantastically, including Marc’s win.
"Both victories in the race, this overall win I confirmed today and my first success in 2012, have been really significant into my sporting career and for my preparations for the Tour. Reaching the ‘Grande Boucle’ this way feels really good.”