Statistical experimental design combined with principal component analysis was used to evaluate interdependence of process variables in enzymatic transesterification. The reaction studied was alcoholysis of rapeseed oil methyl ester (biodiesel) and trimethylolpropane. The reaction products are trimethylolpropane esters which can be used as raw materials for biodegradable hydraulic fluid and other lubricants. The methods used revealed some hidden factors that affect the reaction. Water activity and elimination of the by-product methanol were found to be the principal factors affecting the product yield. Mono-, di-, and trisubstituted esters were produced in different optimum conditions. The model developed for monosubstituted ester was verified with a bubble column experiment.