The 19th-seeded Latvian needed just 85 minutes to claim victory, winning 57 percent (67 of 118) of the points and repeatedly converting in key moments.
Williams was on track to meet defending champion Sloane Stephens in the semi-finals but her third-seeded countrywoman was dumped out of the tournament via a 6-2, 6-3 loss to 19th seed Anastasija Sevastova.
She would equal Margaret Court's record for most major titles by winning Saturday. In that match, Williams couldn't overcome a slow start, never recovered her nerves, never did what she was able to do Tuesday night.
Serena looked off her game early on Wednesday (AEST) at Arthur Ashe Stadium in New York City, but it didn't last long.
Sevastova, who avenged last year's quarter-final loss to Stephens in NY to reach her first major semi-final, converted five of her eight break points and saved seven of the nine against her on the Arthur Ashe Stadium court.
Williams managed to race into a 4-0 lead with all momentum now shifted onto the American who was looking to close this match out quickly.
Stephens was ranked 83rd in the world when she upset Sevastova in three sets at the same stage past year, trailing by a break in the third set before winning it in a tiebreaker.
The top seeds continued to tumble during second week of the US Open.
She has been a peerless performer in big matches; the victory over Pliskova was her fourteenth consecutive win in quarterfinal matches at grand slams. "It's (going to be my) first semifinals at (the) U.S. Open".
Del Potro, the 2009 Open champion, staved off all three break points he faced in the match, which featured his first dropped set of the tournament.
As one American star lost in the main draw Tuesday, another of the country's rising stars was on display at the U.S. Open.
Officials had suspended the start of play for both the juniors and wheelchair events as temperatures rose into the 90s.
On the previous three such occasions, he'd lost.
Go back to 2015, and that was another semifinal departure for Williams, whose bid for a calendar-year Grand Slam was shockingly ended by Roberta Vinci. They got into the tournament as alternates when 2016 finalists Pablo Carreno Busta and Guillermo Garcia-Lopez withdrew on opening day.
If Isner wins, he would be the first American in the U.S. Open semifinals since Andy Roddick in 2006.
Hot and humid weather continues to cause problems at the U.S. Open.
The US Open had been a bug-bear for the highly strung, but also highly entertaining Anastasija Sevastova to bear.
Williams has performed wonders already by reaching the Wimbledon final, less than 11 months after the birth of her daughter Alexis Olympia. With all the other top ten seeds now out of the tournament and Williams in ominous form, she is the heavy favourite to defeat whoever makes it through.