A simple, rapid and sensitive method based on dispersive derivatization liquid–liquid microextraction (DDLLME) combined with gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) was developed and validated for the determination of prostate cancer metabolite biomarkers, including sarcosine, alanine, leucine and proline, in human urine samples. Dispersive derivatization using isobutyl chloroformate has been successfully employed to identify the amino acids of interest in ng mL−1 concentrations. Under the optimum experimental conditions, the detection limits of the amino acids were in the range of 0.05–0.1 ng mL−1. The enrichment factor and relative recovery for the target amino acids were in the range of 140–155 and 93.8–106%, respectively. The proposed method showed good linearity (correlation coefficients >0.997), and good intra-day (below 7%) and inter-day precision (below 10%). This protocol provides a rapid, simple, selective and sensitive tool to quantify sarcosine and endogenous urinary metabolite for prostate cancer diagnosis and for a screening test.