A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed and validated for the determination of Tirofiban in biological fluids. Tirofiban in serum samples was extracted and cleaned up by using an automated solid phase extraction method. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode (MRM). A good linear response over the range of 2-200ng/ml was demonstrated. The accuracy for Tirofiban ranged from 94.8 to 110.8% within-day and from 103.0 to 104.7% between-day. The lower limit of quantification was 2ng/ml. This method is suitable for pharmacokinetic studies.