Many clinical trials have shown the effectiveness of combination therapy over monotherapy in diabetes management. Sitagliptin (SG) and metformin (MF) are the most common combinations for type II diabetes management. These drugs were combined into one tablet, called Janumet 50/850 (SG/MF). The pharmaceutical industry constantly demands a rapid, simple, sensitive, and valid analytical method for simultaneously determining drugs in pharmaceutical products. Therefore, this study aims to develop an ultraperformance liquid chromatography method for concurrently estimating metformin and sitagliptin in a short run time by applying the response surface methodology. A Box-Behnken design was implemented to study the influence of three independent factors: aqueous phase concentration in the mobile phase (A
5-15%), mobile phase flow rate (B
0.4-1 mL/min), and ammonium formate buffer strength (C
5-20 mM). The data analysis showed a significant negative effect of the flow rate on the retention time and peak area. The optimized analytical condition was performed with 15% aqueous phase concentration, a flow rate of 0.52 mL/min, and a buffer strength of five mM. The analytical method was valid per the International Conference of Harmonization (ICH) guidelines. SG and MF were separated in a short time run of 2 min. The process was reliable in separating and extracting the drugs from the marketed Janumet tablets at a retention time of 0.73 and 1.36 min for SG and MF, respectively.