Pharmacokinetic and pharmacodynamic assessment of ester-containing drugs can be impacted by hydrolysis of the drugs in plasma samples post blood collection. The impact is different in the plasma of different species. This study evaluated the stability of a prodrug, ketoprofen methylester (KME), in commercially purchased and freshly collected plasma of mouse, rat, dog, cat, pig, sheep, cattle and horse. KME hydrolysis was determined following its incubation in commercially purchased and freshly collected plasma of those species. Different esterase inhibitors were evaluated for prevention of the hydrolysis in rat, dog and pig plasma. KME was rapidly hydrolyzed in both commercially purchased and freshly collected plasma of mouse, rat, and horse. The hydrolysis was initially quick and then limited in cat plasma. KME hydrolysis was minimum in commercially purchased plasma of dog, pig, sheep and cattle but substantial in freshly collected plasma of those species. Different esterase inhibitors showed different effects on the stability of KME in rat, dog and pig plasma. These results indicate that plasma of different species has different hydrolytic activities to estercontaining drugs. The activities in commercially purchased and freshly collected plasma may be different and species-dependent. Esterase inhibitors have different effects on preventing hydrolysis of the ester-containing drugs in the plasma of different species.