We aimed to investigate the performance of the development method for sitagliptin quantification analysis in human plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS) using liquid-liquid extraction (LLE) and Nebivolol as internal standard (IS). The parameters of this presented validation method are selectivity, the lower limit of quantification (LLOQ), linearity, accuracy, precision with five different concentration (LLOQ, Low QC, Medium QC, High QC, Upper Limit Of Quantification (ULOQ)), Integrity of dilution, matrix effect, and test for stability. Based on this study, the multiple reaction monitoring (MRM) transitions were m/z 408,23 → 127,02 for Sitagliptin and m/z 406,25 → 151,06 for IS Nebivolol. The selectivity test obtained % interference of sitagliptin and IS nebivolol by (0.00 - 0.38) % and (0.04 - 0.24) %, respectively. LLOQ test obtained a concentration value of 10.32 ng/mL and % RSD (n = 5) of 5.27 %. When the Sitagliptin concentration ranged from 1 to 1000 ng/mL, the method showed strong linearity with a coefficient of correlation of 0.9991. Accuracy test obtained % differentiation of (-12.58 - 7.77) % and precision test obtained % RSD of (1.62 - 5.32) %. The dilution integrity test obtained % differentiation at 4 and 2 times dilution was -9.20 % and -9.16 %. Matrix effect and stability data were in line with the stipulated European Medicines Agency (EMA) guidelines for validating the bioanalytical method.