The synthetic substrate cholyl-PABA, developed by conjugating cholic acid with paraaminobenzoic acid, is hydrolyzed by the bacterial enzyme cholyl hydrolase to release free PABA. This study aimed to evaluate whether quantitating urinary excretion of PABA after oral administration of cholyl-PABA can detect small intestinal bacterial overgrowth. In the first phase, investigations were performed on 10 healthy volunteers to study the dynamics of urinary excretion of PABA and any adverse reactions after oral administration of 1.2 g of cholyl-PABA. Another 10 healthy volunteers and 25 adult patients with various gastrointestinal disorders participated in the second phase, where the urinary cholyl-PABA test was compared to the [14C]xylose breath test (XBT). The upper limit of normal levels of urinary PABA excretion at the end of 4 h was 1.1% of the administered dose of cholyl-PABA. The urinary PABA excretion after 4 hr [median (range), in percentage] in the XBT-positive group was 1.6 (0.6-35.0), which was significantly higher than those in the XBT-negative group [0.7 (0.4-1.8)] and the healthy controls [0.7 (0.2-1.1)]. The agreement between the XBT and the urinary cholyl-PABA test was 85.7% (P < 0.01). No adverse effect was noted. In conclusion, the urinary cholyl-PABA test offers a simple, safe, noninvasive, and rapid method for diagnosing small intestinal bacterial overgrowth and warrants further clinical evaluation.