Lipase hydrolyses triglycerides to release free fatty acids and glycerol.

                 Triglycerides + H₂O → Glycerol + Fatty acid

The assay of lipase activity is based on the method of hydrolysis of triglycerides in olive oil by lipase into fatty acids, diglycerides and to some small extent into mono glycerides and glycerol.

                Olive oil + Lipase → Fatty acid + Diglycerides

The amount of fatty acids formed, under the specific conditions of the test, is a measure of lipase activity in the sample.

The fatty acids formed are determined by titration with sodium hydroxide.

Sodium azide has been added as a bacteriostatic agent to the substrate without affecting results.