The development of the bio analytical techniques brought a progressive discipline for which the future holds many exciting opportunities to further improvement. The main impact of bio analysis in the pharmaceutical industry is to obtain a quantitative measure of the drug and its metabolites. The purpose is to perform the pharmacokinetics, toxic kinetics, and bioequivalence and exposure response like pharmacokinetic/pharma co-dynamic studies. Various bio analytical techniques are performed in bio analytical studies such as hyphenated techniques, chromatographic techniques, and ligand binding assays. This review extensively highlights the role of bio analytical techniques and hyphenated instruments in assessing the bio analysis of the drugs.