Bioanalytical methods are used to create a protocol that can identify and quantify bioactive molecules and their metabolites in human and animal samples. For determining the medication and its metabolites present in human/animal body, the bioanalytical method is efficient. One of the prominent bioanalytical roles is creating new techniques, validating existing processes, and analyzing samples. Most importantly, a compound can be calculated using many methods and can also be detected using various analytical methods. In specific biological samples, the drugs can be evaluated by different extraction procedures, such as solid-phase extraction, liquid extraction, protein precipitation etc.., All the steps in methods were investigated to know how environment, sample, or techniques influence the matrix assessment up to the study's time. The analytical methods, including high-pressure (HPLC), liquid chromatography combined with dual-mass spectroscopy (LCMS-MS), and Ultra-performance extraction (UPLC), can be used to obtain the in-depth analysis of medications. All have their strengths and weaknesses. Currently, bioanalysis is usually done by HPLC-MS and GCMS-MS. Linearity, repeatability, Accuracy, precision, selectivity, recovery and consistency are essential criteria. In this review we proposed to give importance of bioanalytical system production and validation parameters to aid in drug quality, purity, safety and efficacy.