Metabolites impart a significant importance to the understanding of biological reactions and consequently to the development of diagnostic and therapeutic techniques for specific diseases. Furthermore, there has been recent interest in metabolite concentrations present in urine for potential noninvasive disease diagnosis. The detection of specific metabolites, however, presents certain analytical difficulties such as low or ambiguous specificity of the techniques. This study developed a new technique, utilizing oxidative, enzymatic production of formaldehyde from the metabolite to produce a pH-induced change observed by fluorescein in acetone. This probe displays high sensitivity towards pH imbalances and, coupled with high enzymatic specificity, forms an accurate method to measure metabolite concentrations. Sarcosine was used as a model analyte in this study due to its potential for serving as a prostate cancer biomarker. Sarcosine was treated with sarcosine oxidase to generate formaldehyde, which was further oxidized to formic acid, and subsequently measured by the corresponding change in fluorescein. A good linearity was revealed with a correlation coefficient of 0.9961 and a detection limit of 20 nmol L⁻¹. This method was applied to sarcosine analysis in nine urine samples. The results suggest that this is a viable, cost-effective technique for determination of sarcosine in urine samples without interferences such as alanine.