Pruitt, in appearances before congressional panels this spring, has repeatedly blamed subordinates for his alleged ethical lapses.
Pruitt's statement called Hupp "a valued member of the EPA team".
EPA Administrator Scott Pruitt is mired in scandals.
Pruitt, the former attorney general of Oklahoma, suggested the unending stream of news reports about him and new federal investigations were political.
"I think with great change comes, I think you know, opposition".
Democratic Sens. Tom Udall of New Mexico, Tom Carper of DE and Sheldon Whitehouse of Rhode Island asked EPA inspector general Arthur Elkins on Thursday to investigate whether Pruitt is illegally using his EPA post to drum up business deals for his family.
She spearheaded his apartment search when his lobbyist landlords soured on his continued presence in their Capitol Hill condo; booked his personal travel; and inquired with President Donald Trump's DC hotel - located across the street from EPA - about purchasing a used mattress Pruitt had heard was available.
He also says his wife is an entrepreneur, as if somehow that makes breaking the law OK.
NASA finds more stuff suggesting Mars could have hosted life, maybe
The Curiosity rover has not been able to find the source of the organic molecules in its findings, according to the report. They're considered necessary for life to form , but there are plenty of places with lots of organic compounds but no life.
A Chick-fil-A representative told The Washington Post that Pruitt was interested in his wife becoming a franchise owner, but that she didn't complete the application.
On Wednesday, Pruitt laughed when a reporter asked about the reports he had tried to use his government position to financially benefit his spouse.
She becomes the second top Pruitt aide in two days to resign.
Rep. Elijah Cummings of Maryland, the ranking Democrat on the House Oversight committee investigating Pruitt, said Hupp shouldn't have to take the fall for her boss.
It caps off a week full of reports about Pruitt amid further scrutiny into his tenure at the EPA.
In her testimony provided the House Oversight Committee, Hupp said she spent several hours a week at work discussing housing accommodations for Pruitt with realtors and visited at least 10 properties on behalf of Pruitt, who asked her verbally to assist him in finding housing options, according to the testimony.
For his part, Pruitt suggested the criticism, investigations and unending stream of negative revelations about him were all political attacks motivated by his efforts to roll back environmental regulations.
"I just know there's a lot of talk of conflicts and we'll see how much the market can bear", he said.