Yovanovitch made no comment to the press as she arrived at around 10:15 a.m. local time for a closed-door deposition before the US House Intelligence Committee.
The career diplomat, who joined the US Foreign Service in 1986, was abruptly removed from her post in May. Her ouster was one of several incidents listed in a whistle-blower complaint filed by a CIA officer alleging that Trump withheld foreign assistance from Ukraine in order to pressure the country into investigating his political rival, former Vice President Joe Biden.
The whistle-blower complaint has spawned a House impeachment enquiry into whether Trump abused the power of his office to solicit foreign interference in a US election.
The complaint alleged that Trump during a July 25 phone call pressed Ukraine's President Volodymyr Zelenskyy to investigate Biden.
Trump denies any wrongdoing and insists that the phone call with his foreign counterpart was "perfect."
During his conversation with Zelinsky, Trump called Yovanovitch "bad news," according to the transcript of the call released by the White House.
Trump's allies have accused Yovanovictch of working to undermine the US president's agenda. In an interview with the Wall Street Journal in September, Trump's lawyer Rudy Giuliani said that in the run-up to Yovanovitch's removal, he reminded Trump of complaints about her political bias.
On Thursday, US federal prosecutors unsealed an indictment against two of Giuliani's associates who allegedly engaged in lobbying efforts on behalf of a Ukrainian politician to press for Yovanovictch's ouster.
The indictment accused Lev Parnas and Igor Furman - both US citizens who Giuliani has said were his clients - and two others of funnelling $1 million in illegal donations from foreign nationals to unspecified political campaigns.
In one of several schemes they allegedly carried out, the four men donated money to a member of Congress and then tried to press that lawmaker to "remove or recall the then-US ambassador to Ukraine." That effort, the indictment alleged, was carried out at least partly at the request of "one or more Ukrainian government officials."
Parnas and Furman were arrested late Wednesday at Dulles International Airport while waiting to board an international flight out of the United States with one-way tickets.