Current in vitro pancreatic lipase inhibitor screenings are based on previous spectrophotometric lipase assays. Nevertheless, they are with little evaluation of assay conditions. This study focuses on the impacts of experimental factors on enzyme activity in the assay with p-nitrophenyl palmitate as substrate by monitoring their effects on the hydrolysis rates. On the results, experimental conditions for lipase inhibitory assay were proposed. Notably, 5 mM sodium deoxycholate as emulsifier not only maintains the assay homogeneity but also enhances lipase activity. Organic co-solvents to dissolve organic inhibitors including DMSO, EtOH, MeOH, IPA, AcCN 0–30% (v/v) was found well tolerated by the enzyme. With 10% (v/v) glycerol, lipase solutions can be stored at –20°C for up to one month without significant loss of activity. The results reported here provide researchers the assay condition sets in which most inhibitors can be dissolved, and lipase activity is not severely affected. This could accelerate the rational development of novel lipase inhibitors.