The development of novel techniques for sample collection, preparation, bio analytical methods, and method validation is necessary to meet the pharmaceutical industries' demands for high throughput analysis and screening. It is very desirable to use bio analytical methods for drug research and discovery as well as impurity profiling to preserve the efficacy, safety, and quality of medications. This review searches for new research on the validation of bio analytical techniques and the creation of bio analytical technique employing biological fluids such as plasma, serum, urine, saliva etc. We also keep an eye out for research that addresses the many methods of sample preparation that are employed in bio analysis. Any analytical method’s validation aids in producing accurate data, which are essential for making wise judgments regarding patient safety and medication dosage. Sample preparation and method validation are required to demonstrate the efficacy of the procedure and the analytical results. Nowadays, it is generally recognized that bio analysis is essential to the PK (pharmacokinetic) and PD (pharmacodynamics) assessment of a new chemical entity, starting from the periods of discovery and continuing through the many stages of development of drugs that lead to the drug’s approval for commercialization. This study systematically assesses significant bio analytical validation criteria, including accuracy, precision, sensitivity, selectivity, quantification limits, range and recovery. This review also discusses the latest FDA (Food and Drug Administration) standards for sample preparation methods and chemical extraction from different biological matrices. The practical and regulatory perspectives that researchers may employ to develop and evaluate the bio analytical technique are the main emphasis of the current study.