Carboxylesterases are important enzymes for xenobiotic metabolism and are receiving increasing attention in the context of cancer therapies. Quantification of individual carboxylesterase activity is important since protein levels do not always correlate to activity and significant interorgan, interindividual, and interspecies variations exist. Here we present a methodology enabling the specific quantification of carboxylesterase 2 activity in a pool of other esterases. Method applicability is illustrated for the evaluation of interspecies variation and for activity assessment of transfected cell extracts. The methodology can easily be adapted to the evaluation of other esterases upon careful selection of adequate substrates and/or specific inhibitors.