For a study, it was determined that excessive hydrogen sulfide (H2S) production from gut microbial metabolism could play a role in the etiology of gastrointestinal illnesses including ulcerative colitis. However, little was known about the variables that influence its development. The aim of the research was to verify real-time H2S measurement repeatability and then assess its generation following exposure to dietary variables and 5-aminosalicylate acid using a newly constructed in vitro gas-profiling system (5-ASA). H2S, carbon dioxide, hydrogen, and methane readings were compared using different gas profiling devices. Freshly passed healthy human feces were used to make homogenized slurries. About 50 mL slurries were aliquoted into separate fermentation chambers, and substrates such as 1g highly fermentable fructooligosaccharides (FOS) or resistant starch Hi-Maize (RS), or minimally fermentable psyllium or sterculia, 1g cysteine, 0.9g sodium sulfate, or 1.2mL of 1 M 5-ASA have added alone or in combination. Over the course of four hours, H2S emission was measured every five minutes and compared to unspiked controls. RS reduced H2S and FOS production by an average of 89.0(SEM 4.8)% and 82.2(6.2)%, respectively, compared to <35(17)% and 82.2(6.2)%, respectively, for psyllium and sterculia (p<0.001, two-way ANOVA). H2S generation was increased by 1557 (532)% when cysteine was added. In comparison to the addition of 5-ASA (0.3(2)%, p<0.001) to slurries containing cysteine, fructooligosaccharides (FOS) dramatically lowered H2S by 90(2)%. Overall, the effects of sulfate and 5-ASA were minor. Finally, the H2S-profiling technology was a repeatable method. Sulfur-amino acids, but not inorganic sulfate, considerably increased H2S production, while rapidly fermentable fibers efficiently decrease it. The findings could help in the development of dietary therapy to lower H2S generation in humans.