A selective, sensitive, stable and rugged high performance liquid chromatography tandem mass spectrometric method was developed and validated for determination Quetiapine in human plasma, then successfully applied in pharmacokinetic study for two different tablet doses of quetiapine (50 and 200) mg. The mobile phase consisting of (70% methanol: 30% water), pH=3.0. The method was validated according to European guideline for its linearity, accuracy, precision, selectivity, recovery and various stabilities. The standard curve was linear over a concentration range of (2.25-500.0) ng/ml, R2 was 0.999 and CV% of intraday accuracy and precision for the validation of QC samples was ranged between (1.82-3.61) %. A randomized, double-blind, two periods crossover full study was designed to investigate two different tablet doses from Quetiapine (50 and 200) mg. 40 healthy male volunteers were participated in this study, and the oral administration was occurred after overnight fasting. Blood samples were withdrawn from each subject at different time intervals and analyzed for Quetiapine concentrations. The pharmacokinetic parameters were performed and show that the maximum time of absorption (T max) for both doses was the same at 4.5 hr. The maximal plasma concentrations were (49.058 and 262.355) ng/ml and the Kel (0.07993 and 0.09207) for the (50 and 200) mg doses, respectively. However, based on these statistical data, quetiapine low and high doses were well tolerated and an obvious significant variation (p value less than 0.05) was found between them in human plasma.