In the present study, several key reaction parameters that affect the hydrolytic rate of the p-NP palmitate by Thermomyces lanuginosus lipase were studied to define new spectrophotometric assay for measuring the lipase activity. The influence of the substrate and enzyme concentration, reaction time, pH, organic solvents (acetonitrile, methanol, isopropanol, ethanol, 2-ethoxyethanol, DMSO, acetone, and 1,4-dioxane) and emulsifier agents (Gum Arabic, PEG 4000, PEG 6000, Triton X-45, Triton X-100, SDS, CTAB, Tween 20, Tween 40, Tween 80, and Span 80) on the reaction rate was analyzed. Two kinetic models, Michaelis–Menten kinetic model and Hill kinetic model were suggested. The results obtained by developed models were compared, and it was shown that Hill kinetic model describes experimental data better. A spectrophotometric test is designed for its primary applications for the measuring of lipase activity during the enzymatic biodiesel production process and for the measurement of lipase activity during its production.