The two rivals are vying to emerge as the chief alternative to the absent former president Donald Trump, who has maintained a commanding lead in opinion polls ahead of Iowa's January 15 contest.
The debate on Wednesday night, which also included tech entrepreneur Vivek Ramaswamy and former New Jersey Governor Chris Christie, saw a flurry of insults interspersed with discussions of the Ukraine war, the Israel-Hamas conflict and the US southern border.
But aside from Christie, who has put criticisms of Trump at the centre of his campaign, none of the candidates on stage in Tuscaloosa, Alabama, appeared willing to go after the frontrunner directly, a reflection of Trump's continued popularity among the Republican base.
Asked about Trump's comments at a Fox News town hall on Tuesday that he would not be a dictator during a second term, except on "day one," Christie called him "an angry, bitter man" and repeatedly demanded DeSantis say whether he believes Trump is fit for office.
DeSantis deflected several times, instead referring to the 77-year-old Trump's age and arguing the presidency is better suited for someone younger.
Haley also offered only muted criticism, blaming Trump for adding billions of dollars to the national debt.
Instead, DeSantis and Ramaswamy spent most of their time taking shots at Haley, who has risen in polls and drawn increased interest from donors on the strength of her previous debate performances.
"She caves anytime the left comes after her, anytime the media comes after her," DeSantis said of Haley during the first answer of the evening, as he sought to explain why voters should back him despite Trump's dominant position.
DeSantis boasted about legislation he passed in Florida banning gender-affirming medical care for transgender youth and accused Haley of opposing the law, an assertion Haley denied.
"He continues to lie about my record," she said.
As with the first three debates, the former president - leading by more than 40 percentage points in most opinion polls - skipped Wednesday's event, instead attending a fundraiser in his home state of Florida.
Trump's absence deprived his rivals of an opportunity to confront him face to face and again sent the message he deems his challengers unworthy of his attention.
DeSantis holds a small but shrinking advantage over Haley in national polls, while Haley has a substantial edge over DeSantis in New Hampshire and her home state of South Carolina - crucial states because they are among the first to pick a nominee.
Ramaswamy, a staunch isolationist, was alone in arguing the US should end its support for Ukraine against Russia. He took aim at Haley, who has emphasised her foreign policy experience, saying that experience "is not the same as wisdom".
In response, Christie defended Haley, telling Ramaswamy he was coming across as "the most obnoxious blowhard in America."
Meanwhile, Haley, who called Ramaswamy "scum" at the last debate, said it was "not worth my time to respond" after he again called her corrupt.
Ramaswamy also advanced a number of baseless conspiracy theories, claiming the January 6, 2021, assault on the Capitol by Trump supporters was an "inside job" and that the 2020 election was stolen.
Only Christie appeared willing to attack Trump directly, criticising him for saying during a Fox News town hall on Tuesday that he would not become a dictator except on "day one".
"I've got these three guys who are all seemingly to compete with, you know, Voldemort, he who shall not be named," Christie said, referring to the unspeakable villain in the Harry Potter stories.
"They don't want to talk about it."
