Deputy Chief Whip John Randall and Cabinet Office Minister Chloe Smith both announced they would be standing down last night and returning to the back benches.
Both resignations come ahead of a Cabinet reshuffle which Prime Minister David Cameron is expected to carry out in the coming weeks.
Mr Randall, the MP for Uxbridge and South Ruislip since 1997, was responsible for maintaining party discipline among Tory MPs.
He remains a central figure in the sequence of events that saw his old boss, the former Chief Whip Andrew Mitchell, stand down in September last year amid accusations he had insulted police officers who refused to allow him to ride his bike through a gate at Downing Street.
Randall alerted No 10 to a police officer's account of Mr Mitchell's confrontation with armed protection during which he allegedly called them "plebs".
He then also threatened to quit in March last year unless Mr Mitchell, with whom he is said to have a strained relationship, left his post.
Mr Mitchell continues to protest his innocence in the row and two men have been arrested amid an ongoing police investigation into the claims.
In his resignation letter, Mr Randall, 58, said it had been "a great privilege and honour" to serve for 13 years in the Whips' office in opposition and in government.
Ms Smith tweeted that her constituency work had "always mattered most" after she announced her resignation on her personal blog
The 31-year-old, the youngest MP in the House of Commons, said she had written to David Cameron last month to say she would be leaving her post when he next reshuffled the Cabinet.
She said: "I am proud to have served my country in this way but my constituents have always come first for me.
"They know the high standards and the hard work that I ask of myself as their MP. As an elected Conservative Member of Parliament, the Prime Minister and my party have my full support as we continue to fix the economy, reward hard work and do the right thing for Britain."
Ms Smith hit the headlines last year for her stumbling performance during a live interview with Jeremy Paxman on Newsnight after the Treasury decided to delay a planned 3p rise in fuel duty in August. The MP, at that time a junior Treasury minister, was subjected to a gruesome mauling at the hands of the veteran broadcaster after she appeared unable to say when she had learned of the decision.