The lipase from Mucor javanicus was shown to catalyze the acylation of the primary hydroxyl (C14-OH) of doxorubicin (DOX), a potent anticancer compound. An ion-pairing method for solubilizing enzymes in organic solvents with the anionic surfactant Aerosol OT (AOT) was then adapted to enhance the non-aqueous activity of the lipase, representing the first demonstration of this solubilization and activation technique for a lipase. The pH and ionic strength of the aqueous phase during solubilization were identified as the factors having the greatest impact on the extraction efficiency and specific activity of the biocatalyst. A series of expanding experimental matrices yielded both solubility and specific activity surfaces as functions of [NaCl] and pH in the extraction of M. javanicus lipase. The activity response surfaces were generated with the esterification of octanoic acid with 1-nonanol in isooctane as a convenient model reaction, yet the results were shown to transfer to the acylation of DOX with 2-thiophene acetic acid vinyl ester, or vinyl butyrate, in toluene. A generalized approach to ion-paired lipase solubilization was thus developed, and a potentially high-value biotransformation was enhanced using a low-cost and easily-assayed reaction.