[30] Robustness It is the measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters and provides an indication of its reliability during normal usage. Extraction recovery It can be calculated by comparison of the analyte response after sample workup with the response of a solution containing the analyte at the theoretical maximum concentration. Therefore, absolute recoveries can usually not be determined if the sample workup includes a derivatization step, as the derivatives are usually not available as reference substances. Stability It is the chemical stability of an analyte in a given matrix under specific conditions for given time intervals.[30] The aim of a stability test is to detect any degradation of the analyte(s) of interest during the entire period of sample collection, processing, storing, preparing, and analysis.[39] All but long-term stability studies can be performed during the validation of the analytical method. Long-term stability studies might not be complete for several years after clinical trials begin. The condition under which the stability is determined is largely dependent on the nature of the analyte, the biological matrix, and the anticipated time period of storage (before analysis). The ICH guidelines are summarized in Table 1. Table 1 US FDA guidelines for bioanalytical method validation The drug research can be divided functionally into two stages: discovery/design and development [Figure 1]. Figure 1 Different stages of discovery/design and development DRUG DISCOVERY/DESIGN Initially, in the discovery stage, the aim of bioanalysis could be merely to provide reasonable values of either concentrations and/or exposure which would be used to form a scientific basis for lead series identification and/or discrimination amongst several lead candidates. Therefore, the aim of the analyst at this stage should be to develop a simple, rapid assay with significant throughput to act as a great screening tool for reporting some predefined parameters of several lead contenders across all the various chemical scaffolds. The initial method of analysis developed during the discovery phase of the molecule, with some modifications, may sometimes serve as a method of choice to begin with as the NCE enters the preclinical development stage. Since the complexity of development generally tends to increase as the lead candidate enters the toxicological and clinical phase of testing, it naturally calls for improved methods of analytical quantization, improvement in selectivity and specificity, and employment of sound and rugged validation tools to enable estimation of PK parameters that would also aid in the decision-making of the drug molecule’s advancement in the clinic in addition to safety and tolerability data gathered at all phases of development. Additionally, it becomes necessary to quantify active metabolite(s) in both animals and humans.