A prodrug is a pharmacologically inactive derivative of an active parent drug, and it that is bioconverted to the active drug in vivo. Through the chemical modification of a drug to a prodrug, we are able to deliver drugs into to the target site, to optimize therapy and minimize toxicity. A major pathway for the bioconversion of prodrugs to the active parent drugs is via carboxylesterase (CES) activity. Among human CES isozymes, hCES1 and hCE2 predominantly participate in the hydrolysis of prodrugs in the liver and small intestine, respectively, although the substrate specificity is quite different between two isozymes. Since the expression levels of CES vary among individuals, there is a range of pharmacological responses following prodrug administration. Species differences are caused by the tissue-dependent hydrolase activity mediated by CES, which makes it difficult to predict effectiveness in humans from a preclinical study using animals. The hydrolysis parameter of several ester prodrugs in the in situ rat jejunal single pass perfusion has been related to the in vitro hydrolysis parameter in the intestinal S9, in order to propose the noble quantitative prediction of intestinal first pass metabolism by in vitro-in situ correlation. We have developed a novel experimental method for predicting the human intestinal absorption of prodrugs using Caco-2 cells in which CES-mediated hydrolysis has been inhibited. The expression of hCE1 and hCE2 shows inter-individual variation and is regulated by several mechanisms, such as gene polymorphism and epigenetic processes. Understanding of the regulation of CES expression and species difference of CES catalytic properties will be helpful in the design of prodrugs with increased specificity and enhanced physicochemical and biological properties.