NEW YORK, NY, USA - Czech qualifier Karolina Muchova pulled off the biggest win of her young career well after midnight on Thursday at the US Open, outlasting two-time Grand Slam champion Garbiñe Muguruza 3-6, 6-4, 6-4, to win just her second match in a Grand Slam main draw.

The 22-year-old made her Grand Slam main draw debut in Flushing Meadows by winning three rounds in qualifying, and put on a fearless display to unsettle and ultimately upset the former World No.1 after two hours and 27 minutes on Louis Armstrong Stadium.

"It's unreal, it's perfect. I can't be better," she exclaimed after the win. "I don't think I've realised what I've done yet.

"It was new for me, playing at night and under the lights. I made a lot of mistakes and then I got used to it.

"I wanted to play my game, keep her back and I think it worked out."

Things began brightly enough for Muguruza, who ran off the first five games of the match. Muchova soon settled, however, and got herself into the match, breaking the Spaniard as she served for the set and clawed back to 5-3. The 2017 Wimbledon winner managed to close it out on her second opportunity and get within a set of the third round.

Things got more complicated in the second set as the No.12 seed fell behind 1-4 only to level proceedings by winning the next three games. Serving to stay in the set, Muguruza found herself outfoxed at the net as Muchova engineered a set point. The youngster practically caught fire chasing down a forehand and smacking it down the line to level the match.

"I could breath in the second set and then I got into it," she admitted. "I just played my shots."

Muguruza looked to have steadied early on in the decider, edging ahead 2-0, only for Muchova to respond with three straight games of her own. Tense games followed as the set continued on serve, Muchova pressing and charging the net while Muguruza struggled to find a consistent rhythm off the ground.

Muchova held to love to get within a game of victory, and struck a stunning pair of winners - one forehand, one backhand - to earn match point. A net cord disrupted the Czech's flow as she put a backhand into the net, but a forehand error from Muguruza quickly pulled up a second match point.

The qualifier made no mistake this time, putting in a deep return that the Spaniard pulled long to secure the career-defining upset.

"I think she played very well," Muguruza admitted. "My game didn't affect her. My level wasn't there. 

"I've never had a summer with so few matches. When I play tough matches, I need to have a feeling I've done it in the past. She played better."

Up next for Muchova is No.18 seed Ashleigh Barty, who cut through the noise to knock out former French Open finalist Lucie Safarova, 7-5, 6-3.

"I don't know her," Muchova admitted. "I'll watch her on the internet with my coach."