Purpose: Multiple protocols exist for the dosing and duration of human chorionic gonadotropin (hCG) and the sampling schedule of the hCG stimulation test. This study analyzes the testosterone response following a single-dose hCG test. Methods: This observational study analyzes 103 prepubertal undervirilized males who underwent a single-dose hCG test. A dosage of 1,500 IU was used for those under 2 years of age and 5,000 IU for those over. Testosterone levels were measured before and 72 hours after the hCG injection, and the fold of increase was analyzed. As part of the unit protocol, those with a suboptimal response to a single-dose test (n=19) underwent a 3-day hCG test. Results: A significant 23.65-fold increment of testosterone, with a poststimulated value of 167.26 (interquartile range [IQR], 62.30–279.15) ng/dL, was observed following a single dose of hCG. Of the 103 subjects, 19 (18.4%) had a subnormal response with testosterone levels of 8.20 (IQR, 3.48–29.70) ng/dL. A 3-day test on these 19 subjects showed a testosterone level of 18.4 (IQR, 10.6–64.2) ng/dL, which is statistically significant. However, the 3-day hCG test revealed an adequate response in only 3 patients. The remaining 16 did not achieve the expected outcome, and 15 of these patients had laboratory evidence of hypogonadism either genetically or biochemically. Conclusion A single-dose hCG stimulation test could serve as an alternative to a 3-day hCG test in the initial assessment of Leydig cell function, thereby avoiding repeated injections, hospital visits, and school absenteeism.