The analysis of endogenous and exogenous analytes in biological matrices presents several challenges to the bioanalyst. These analytes are often present at low concentrations, typically in complex matrices, and may have physicochemical properties that are not amenable to LC-MS analysis. The bioanalyst thus relies heavily on the formation of analyte derivatives for the efficient quantification of these compounds. These derivatives are also critically employed to derive information on the biology of living systems, potential drug or disease targets, and biomarkers of drug efficacy, safety, or disease progression. In this perspective, we demonstrate how analyte derivatives are applied in modern bioanalytical workflows and we discuss the potential use of these derivatives in the future.