Ibuprofen was chosen as a test compound to perform a multivariate design in order to determine the highest yield of thein vitro glucuronidation reaction in relation to the concentration of reacting and activating substances. Preliminary studies with a univariate design indicated that the concentration of Mg²⁺ had no significant effect on the glucuronidation and that Triton X-100 could be omitted as it appeared to inhibit the glucuronidation. In a 3³ factorial design the influence of concentrations of ibuprofen, UDP glucuronic acid and enzyme, respectively, on the yield of ibuprofen glucuronide was established. It was concluded that the highest amount of ibuprofen glucuronide formed (within the limits of this design) was achieved with an ibuprofen concentration of 486 M, a UDP-glucuronic acid concentration of 3 mM and an enzyme concentration of 3.57 mg/ml. Using this methodology it is possible to optimize the glucuronidation yield in a more rational way, which can be useful in the upscaling of enzymatic reactions.