Introduction: Saquinavir is the first protease inhibitor. Proteases are enzymes that cleave protein molecules into smaller fragments. No analytical procedure was reported for the estimation of saquinavir by LC-MS/MS. Aims: The objective of the present research was to develop a specific liquid chromatography-electrospray ionization tandem mass spectrometric technique for the quantitation of saquinavir in biological matrices. Methodology: Chromatographic elution was attained thru a Thermo Hypersil ODS stationary column having the dimensions of 50 __ampersandsigntimes; 4.6 mm and particle size of 2.4 __ampersandsignmu;m. Isocratic elution was processed with methanol and 0.1%V/V formic acid in the ratio of 90:10 V/V as mobile phase with flow rate of 0.50 ml/min. Liquid-liquid extraction was performed for drug and internal standard isolation with an ethyl acetate solvent. Parent and productions were monitored at m/z 671.3 __ampersandsignrarr; 654.3 for saquinavir and 614.3 __ampersandsignrarr; 596.3 for indinavir internal standardon multiple reaction monitoring. Result: Linearity graph of drug was rectilinear in concentration over 260.4. to 10416ng/ml having r2(correlation coefficient) value more than 0.999. Percentage of RSD findings were __ampersandsignle;5.3% for inter and intraday accuracy and precision. This procedure has good recoveries and %recovery findings of lower quality control (LQC), median quality control (MQC) and higher quality control (HQC) samples were 94.6%, 92.4 and 104.2% respectively. Conclusion: Saquinavir has more stability for longer time when subjected for different stability environments and the technique was effectively relevant to routine analysis of saquinavir in biological matrix.