Alogliptin benzoate (ALG) is a dipeptidyl peptidase-4 inhibitor used to treat type 2 diabetes mellitus. In this study, a spectrofuorimetric method was developed for the derivatization of ALG to develop a fuorescent derivative. The derivatization reaction was carried out by reacting ALG with ninhydrin and phenylacetaldehyde in Teorell and Stenhagen bufer at pH 6.6. The reaction product was a fuorescent pyrrolidone derivative that was observed at λex of 385 nm and λem of 475 nm. The efects of various experimental conditions on the derivatization reaction were investigated. The optimal conditions were found to be a reaction time of 15 min, a temperature of 80 °C, and a concentration of ninhydrin and phenylacetaldehyde of 1.4 mg mL−1 and 0.028% v/v, respectively. A calibration plot was constructed in the concentration range of 20–460 ng mL−1. The calibration curve had a high correlation coefcient of 0.9991 and a low detection limit of 6.57 ng mL−1. The eco-scale penalty points for the derivatization method were calculated to be 86. This indicates that the method is environmentally friendly and has a low risk of generating harmful waste. The derivatization method described in this study provides a sensitive and green method for the quantifcation of ALG in pure form, dosage form, spiked and real rat plasma.