The fluorescence characteristics of sitagliptin phosphate were used to develop a methodology that allowed its determination in pharmaceutical formulations and urine samples; under the studied conditions, limits of determination and quantification of 0.25 and, respectively, 0.85 mg/L were achieved. Linear correlation between fluorescence analytical signal and sitagliptin concentration was achieved up to 10.0 mg/L. The method was considered selective for sitagliptin determination in pharmaceutical formulations because no interferences due to excipients present in considered matrix were observed (as demonstrated by recovery tests comparing analytical and addition curves). When the method was applied to urine samples, Interferences related to the matrix were observed, which made a solid-phase extraction system necessary. The use of calibration was possible only by applying the standard addition method.