Drug dosing for children is frequently suboptimal when relying on the traditional approach of normalizing doses based on body weight or surface area. Thus, the ontogeny profiling of drug-metabolizing enzymes (DMEs) is important to help develop physiologically-based pharmacokinetic (PBPK) models for predicting safer pediatric dosing. In particular interest are hydrolases are one of the most diverse classes of DMEs and catalyze reactions containing esters, amides, phosphates, etc. in both drugs and prodrugs. Hydrolases such as carboxyl esterases, cathepsins, and arylacetamide deacetylase have been studied for their involvement in drug metabolism. However, a significant number of hydrolases have not yet been appropriately characterized, and as such their influence on drug metabolism remains unknown. Moreover, there is a lack of comprehensive data on the ontogeny and inter-individual variability in expression of these enzymes. Here a characterization of the age-dependent protein abundance of hydrolases in viable primary human hepatocytes isolated from pediatric (n=50) and adult (n=8) donors (Female=28; Male=30) using quantitative global proteomics-based total protein approach was performed.