Background <p>Bile acid malabsorption (BAM) is often missed in patients with chronic diarrhea as diagnostic tests are technically challenging and not available widely. Quantitative estimation of fecal bile acids (FBA) in a single stool sample has been reported recently for the diagnosis of BAM and may be easily applied.</p> Methods <p>We performed a pilot observational cross-sectional study to estimate the optimal FBA cut-point for the diagnosis of BAM, using the IDK^® Bile Acid test, an enzymatic spectrophotometry–based assay for measuring total stool bile acids. We estimated FBA concentrations in healthy adults (<i>n</i> = 100; negative controls) and patients with known ileal Crohn’s disease (<i>n</i> = 67; positive controls), generating a receiver-operator characteristics (ROC) curve for assessing its diagnostic accuracy. FBA levels were then assessed in three groups of patients, namely diarrhea-predominant irritable bowel syndrome (IBS-D) and functional diarrhea (FD) (<i>n</i> = 100), post-cholecystectomy (<i>n</i> = 100) and ileal tuberculosis (<i>n</i> = 33).</p> Results <p>Optimal cut-off point for FBA was identified at 2.8&#xa0;µg/g (sensitivity = 89.5%; specificity = 92.0%; area under ROC = 0.959 [95%CI = 0.929–0.989]), with median FBA in healthy controls (1.5 [IQR = 0.7–2.2]) being significantly lower than that in patients with ileal Crohn’s disease (6.0 [IQR = 4.7–8.0]; <i>p</i> &lt; 0.001). Median FBA in patients with IBS-D/FD, post-cholecystectomy and those with ileal tuberculosis were 2.0 (IQR = 1–2.8), 3.4 (IQR = 1.7–5.3) and 3.0 (IQR = 2.2–4.6), respectively. Overall, 21%, 57% and 54.5% of patients with IBS-D/FD, post-cholecystectomy and ileal tuberculosis had BAM.</p> Conclusions <p>We demonstrate the feasibility of quantitative estimation of fecal bile acids in a single stool sample to diagnose BAM.</p> Graphical abstract <p></p>