The lipase inhibitory activity method described here was developed to identify potential anti-obesity properties in milk fermented with different strains of lactic acid bacteria via inhibition of pancreatic lipase and a subsequent decrease in fat digestion and absorption in the gut. The method is based on the hydrolysis of 4-nitrophenyl octanoate by pancreatic lipase and the subsequent release of p-nitrophenol, a coloured product whose absorbance can be measured at 412 nm. Inhibition of lipase leads to a decrease in the amount of p-nitrophenol released and a subsequent reduction in the absorbance with respect to a 100% activity control. The assay was developed by adapting various methods previously described in published literature and includes modifications that are key to adapt the existing protocols to fermented milk samples, in particular the pH issues encountered when analysing acidic samples:•A two buffer system is introduced to allow optimal pH control after addition of fermented milk samples with pH values between 3.5 and 6.5.•A post-clarification filtration step is added for samples where turbidity remains after addition of the clarifying reagent for dairy products.•An absorbance correction factor is calculated and applied to the samples to account for the reduction in the absorbance of p-nitrophenol caused by milk.