Areca nut (AN) is a substance of abuse consumed by millions worldwide, in spite of established oral and systemic toxicities associated with its use. Previous research demonstrates methyl ester alkaloids in the AN, such as arecoline and guvacoline, exhibit mood-altering and toxicological effects. Nonetheless, their metabolism has not been fully elucidated in humans. In the present study, an HPLC-UV bioanalytical method was developed to evaluate the hydrolytic kinetics and clearance rates of arecoline and guvacoline in human liver microsomes (HLM) and cytosol (HLC). The bioassay was capable of quantifying arecoline and guvacoline (and carboxylate metabolites arecaidine and guvacine, respectively) with good sensitivity, accuracy, and precision. Kinetics of arecoline and guvacoline hydrolysis best followed the Michaelis-Menten model. Apparent intrinsic clearance (Cl) of arecoline was 57.8 ml/min/kg in HLM and 11.6 mL/min/kg in HLC, a 5-fold difference. Unexpectedly, guvacoline was dramatically less hydrolyzed than arecoline in both HLM and HLC, with Cl estimates of 0.654 ml/min/kg and 0.466 ml/min/kg, respectively. These results demonstrate, for the first time, arecoline undergoes significant hydrolysis with high clearance rates in the liver. Furthermore, differential tissue metabolic rates and utilization of specific esterase inhibitors unequivocally demonstrated arecoline is a substrate for CES1 and not CES2.