The carboxylesterases (CEs) are hydrolytic enzymes that metabolize xenobiotics that contain ester, thioester, or amide bonds. CEs are ubiquitously expressed but are found in highest concentration in membrane-enriched fractions of the liver. This unit describes assays used to measure the enzymatic activity and tissue distribution of human CEs. Both spectrophotometric and HPLC-based assays are described in detail. Several of these methods are rapid and easy to perform. These methods will be useful to investigators who are interested in studying the metabolism and disposition of esterified drugs and environmental chemicals.