Significant differences in the pharmacodynamic activity and pharmacokinetic properties could exist for a pair of enantiomeric drugs. In order to evaluate the activity, toxicity, absorption, distribution, metabolism, and excretion properties of the individual enantiomers, and any potential for chiral inversion caused by the biotransformation process, chiral bioanalytical assays are necessary for individual enantiomers and/or their metabolites for in vivo samples. However, development and validation of chiral quantitative assays are highly challenging in comparison to typical nonchiral assays. Therefore, a tiered approach should be used to address specific needs arising in different scenarios of chiral drug development, including development of racemate or fixed-ratio (nonracemic) enantiomers, development of a single enantiomer, racemic switches, and quantitation of enantiomeric metabolites. The choice of a nonchiral quantitative assay, a chiral qualitative assay, or a chiral quantitative assay should be based on the development strategy and on the molecular properties of the drug candidate.