Glimepiride and sildenafil citrate are metabolized in liver by cytochrome p450,and there is a possibility of drug interactions if they are taken together. Therefore, asimple and sensitive method for simultaneous determination and validation of sildenafilwith glimepiride in rat plasma has been developed by using high performance liquidchromatography–mass spectrometry (HPLC-MS). A mixture of 70% methanol, 30%of 0.1% formic acid in water was used as a mobile phase, ACE 5 C18 column, theflow rate of 0.5 mL/min was used, the autosampler injection volume was 5 ?L, and clarithromycin was used as an internal standard. The accuracy for glimepiride andsildenafil in all the three days of validation and all the four tested target concentrationwas 99.7% and 98.9% respectively. The standard curves for both drugs matched therequirements, linear relation (R2) ranged between; (0.994 to 1). The serum level ofboth drugs was lowered when combined together, in comparison Serum sildenafil levelwas affected by the combination to a greater extent than glimepiride. Sildenafil wasfound to decrease significantly when it is used in a combination (34.5 ng/mL) comparedto solitary drug use (57.8 ng/mL).