Sharon Smith, whose 5-year-old daughter Tanzie has Down's, confronted Boyle at his stand-up show at the Reading Hexagon after he spent a significant segment of his set mocking people with the disability.
The Mock The Week star spotted Smith and her husband looking uncomfortable in the audience and asked why they were talking during his set. When they told him about their daughter, Boyle tried to laugh off his remarks, asking: "But it's all true, isn't it? Everything I have said is true, isn't it?"
Smith said that she appreciates Boyle is a comedian who trades in dark humour, but claimed that his comments had made her uncomfortable. Boyle responded that it was his last ever tour and said that he "didn't give a f**k". However, he also admitted to the audience that it was the most "excruciating moment" of his career.
After the show, Smith wrote a blog about the events, which has received a swathe of support on social networking sites such as Twitter, with celebrities such as comic actor Mat Horne backing her.
Writing on the blog, Smith said: "My beautiful, feisty, determined 5-year-old daughter has Down's Syndrome and last night Frankie Boyle spent a good few minutes in the second half of his show making joke after joke about people with DS.
"And they weren't even clever or funny jokes either (though the audience were still laughing - well everyone except me and the 5 people I had gone out with last night).
"I know talentless comedians like Jimmy Carr have a history and reputation of poking fun at people with disabilities, but I never expected it from Frankie Boyle. Not at all, indeed I thought he was cleverer than that. I expected dry, nasty, crude humour, yes, but unimaginative humour poking fun at the stereotype of people with Down's Syndrome was not something that I expected."
She continued: "He did not seem to be stopping - jokes about the way people with DS talk, jokes about the way they dress, jokes about the jobs they can do, jokes about their haircuts, jokes about their parents being old and old-fashioned."
Speaking about her confrontation with the comic, she added: "I wanted the ground to swallow me up. I have never felt so small, so stupid, so emotional and so pathetic. How can a stranger make me feel like that?
"I wish that I had managed to explain to them all why I was upset, to tell them how wrong the stereotypes about Down's Syndrome are.
"I love stand up comedians, and I had been so looking forward to seeing Frankie Boyle. I have been to comedy clubs and sat through poor unimaginative comedians poking fun at people with disabilities, and come away unscathed and not needing to start writing a blog about it. But I was disappointed in Frankie Boyle last night. He doesn't need to stoop to such low levels, such poor humour. He is better than that."