Analytical methods play a significant role in the evaluation of drugs and their metabolites in the assessment of pharmacokinetic profile. Since analytical methods and techniques are constantly changing, it is necessary to use well characterized and validated analytical methods to deliver reliable results. Every molecule has its characteristic method of analysis which must be validated based on the long term objective of the analysis. Although, the validation of each of these methods is independent of one another, there are certain conditions that demand the comparison of these methods, such as sample analysis at more than one site using different methods. The comparison of validation of these analytical methods provides inter-laboratory ruggedness. Additionally, it is also essential that the validity of an analytical method be documented before every use such as by constructing a standard curve. However, if the method is used on a regular basis, evidence on its continued validity is routinely provided and repeated validation runs are not required.