Diabetes is one of the most common chronic diseases, and several new oral agents have been approved for type 2 diabetes management in recent years. A combination of different classes of antidiabetic agents is often required to avoid adverse effects such as severe hypoglycemia. A potential novel combination in development involves a fixed dose single-tablet combination of pioglitazone and a dipeptidyl peptidase 4 (DPP-4) inhibitor. DPP-4 inhibitors are a relatively new class of oral antidiabetic drugs. In this study, we simultaneously analyzed eight antidiabetic drugs, including newly developed DPP-4 inhibitors, by high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS). The developed method was applied to analyze the active ingredients in combination drugs and to determine the concentration of antidiabetic drugs in river water. Our results indicate that three recently approved antidiabetic drugs, sitagliptin, alogliptin, and vildagliptin, are present in river water.