We have previously reported on laser spectroscopy as a simple alternative to mass spectrometry. To validate a simplified 13C-urea breath test (UBT) with laser spectroscopy for the detection of Helicobacter pylori in clinical use, we evaluated the optimal time of breath sample collection. The 13C-UBT was carried out on each of 102 infected and 70 non-infected subjects (32 without eradication and 38 after eradication therapy). Breath samples were taken at five time points within 60 min followed by 100 mg of 13C-urea administration. The ratio of 13CO2 to 12CO2 was measured using laser spectroscopy and the recovery of tracer in the exhaled breath was calculated. Results were compared with histological and culture examinations of gastric biopsies to establish the infection status. For statistical evaluation of 13C-UBT, the optimal timing of breath sample collection was examined on the basis of the kinetics of Δ-13CO2. In 32 H. pylori-negative patients (without therapy), the mean ± 2SD of Δ-13CO2 was at its minimum 20 min after urea ingestion whereas in H. pylori-positive patients, the mean ± SD Δ-13CO2 was maximum at 20 min. In addition, receiver operating characteristic (ROC) curve analysis showed that the cut-off value was estimated between 2.5–3.0 per mil (‰) at 20 min before therapy. Based on the histology and culture results, the sensitivity, specificity and positive and negative predictive values were 98.0%, 100%, 100% and 94.1%, respectively. In conclusion, 13C-UBT with laser spectroscopy is a non-invasive, simple, sensitive and specific test to determine H. pylori status. Our findings suggest that in clinical use, measurements made at 20 min after substrate administration could be recommended for most sensitive and specific 13C-UBT results.