A new, specific and sensitive GC-MS method with electron impact ionization technique was developed for quantitative analysis of ezetimibe (EZE) in human plasma. Prior to GC analysis, EZE was derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), which is a trimethyl silylating reagent. The derivatization reaction was optimized and parameters such as catalyst, derivatization time, temperature, solvent and the volume of silylating reagent were investigated. Trimethylsilyl ether derivative of EZE was determined in selected ion monitoring (SIM, mass-to-charge ratio (m/z): 326) mode. The method was validated with respect to LOD and LOQ, precision, accuracy, linearity, specificity, stability, and recovery. The LOQ and LOD were found as 15 and 10 ng/mL, respectively. The linearity of the method ranged from 15 to 250 ng/mL. The correlation coefficient of the calibration curve was 0.9977 +/- 0.0004 (+/- S.E.M.). The intra- and inter-day precisions (RSD) were less than 6% and accuracies (bias) for intra- and inter-day accuracy were found between -4.04 and 9.71 % at four different concentration levels (15, 40, 100, 250 ng/mL). The proposed method was successfully applied to real human plasma samples for determination of total EZE.