A Bioanalytical method utilizes for the quantitative determination of drugs and their metabolites in biological matrix (plasma, urine, saliva, serum etc.) play an important role in estimation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. Development and validation of bioanalytical method is essential to understand the pharmacokinetics of any drug and/or its metabolites. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) is used in laboratories for the qualitative and quantitative estimation of drug substances, drug products and biological samples. This review focus on various extraction techniques like solid phase extraction (SPE), liquid–liquid extraction (LLE), and protein precipitation , which play important role in sample preparation and detection by LC-MS/MS. This article also comprises scanning and optimization of analyte as well as internal standard (IS) and optimization of mobile phase and its preparation. Bioanalytical method validation includes all the parameters like system suitability, sensitivity, selectivity , Matrix Effect, precision and accuracy, calibration curve, stability, Robustness , Ruggedness that demonstrate a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine is reliable and reproducible for the intended use.