The presence of so-called anti-nutritional factors can reduce the bioavailability of nutrients following consumption of seeds which are otherwise an excellent source of proteins, carbohydrates and micronutrients. Among the proteins associated with negative effects on quality in pea (Pisum sativum L.) seeds are lectin, pea albumin 2 (PA2) and trypsin inhibitors (TI). Here we have investigated the impact of these proteins on protein digestibility and amino acid availability, using naturally occurring and derived mutant lines of pea lacking these proteins. The mutations were stacked to generate a triple mutant which was compared with a wild-type progenitor and a line lacking the major seed trypsin inhibitors alone. In vitro digestions following the INFOGEST protocol revealed significant differences in the degree of hydrolysis, protein profile and apparent amino acid availability among the pea variants. Proteins resistant to digestion were identified by MALDI-TOF mass spectrometry and amino acid profiles of digested samples determined. The results indicate that pea seeds lacking certain proteins can be used in the development of novel foods which have improved protein digestibility, and without negative impact on seed protein concentration or yield.