Simvastatin is known as a pro-drug, which could be hydrolyzed by esterases to its active form, simvastatin acid. Although pharmacokinetics of simvastatin and simvastatin acid have been widely studied, hydrolysis of simvastatin to simvastatin acid during blood sampling and plasma preparation has been overlooked in the previous studies, leading to underestimation of simvastatin concentration and overestimation of simvastatin acid concentration in plasma. Since both efficacy and adverse drug reaction of simvastatin are highly dependent on simvastatin and simvastatin acid concentrations in vivo, accurate assessment of the two compounds are critical in their pharmacokinetic and pharmacodynamic studies. The current study was proposed aiming to investigate the esterase mediated hydrolysis of simvastatin in human and rat blood and its impact on the pharmacokinetic study of simvastatin and simvastatin acid. Using various esterase inhibitors including potassium florid (KF), bis(4-nitrophenyl) phosphate (BNPP), and ethylenediaminetetraacetic acid (EDTA), carboxylesterase was found to be the major esterase that hydrolyzed simvastatin in rat blood, while carboxylesterase and paraoxonase were the major esterases mediating the hydrolysis of simvastatin in human blood. Further studies using human recombinant enzymes identified simvastatin as substrates of PON1, CES1b, PON3 and CES1c with Clint of 8.75, 5.77, 3.93, and 2.45 μL/min/mg protein. Therefore, inhibition treatments with 20 mM BNPP and 50 mM KF/ 10 mM EDTA were developed to efficiently prevent the hydrolysis of simvastatin during blood sampling and plasma preparation in rat/human. The subsequent pharmacokinetics of orally administered simvastatin at 8.66 mg/kg in rats found that the Cmax and AUC0-∞ of simvastatin in absence of such esterase inhibitors in the blood sampling process were only 17.04 ± 6.60% and 15.30 ± 6.76% of those in presence of the inhibitors, whereas the Cmax and AUC0-∞ of simvastatin acid were 1.60 ± 0.30 and 1.80 ± 0.22 times of that obtained in presence of the inhibitors. Nevertheless, T1/2 of simvastatin and simvastatin acid remained the same regardless of the blood sampling method. Our current study for the first time demonstrated the importance for assessment of simvastatin stability during the blood sampling and plasma preparation process, which may be applicable to therapeutic drug monitoring of not only simvastatin but also other pro-drugs/compounds sharing similar metabolic properties.