The process of food digestion is a multifaceted physiological phenomenon. In vitro digestion models have been confirmed as valuable substitute models for in vivo digestion in both animals and humans and can be employed as screening instruments to address issues pertaining to food digestion, including digestibility, bioavailability, release of bioactive compounds, and alterations in food structure. Protein hydrolysis stands out as the most significant change in protein digestion, and the extent of protein hydrolysis is commonly considered an indicator of the extent of protein digestion. Despite the acknowledged limitations, the o-phthalaldehyde (OPA) method remains the predominant approach employed by scholars for assessing the extent of hydrolysis. Consequently, the objective of this chapter is to furnish a comprehensive exposition of the aforementioned assay, encompassing a static simulation of the digestive processes and the quantification of protein hydrolysis within a specified sample.