Papaya milk, a mixture of papaya pulp and dairy milk, is one of the most popular beverages in Taiwan. However, the enzymes present in papaya can cause accumulation of hydrophobic amino acids, resulting in a bitter taste of papaya milk. Thus, it is important to select papaya cultivars without the potential to form the bitter taste, but it is difficult to select these papaya cultivars using a sensory test. The purpose of this research was to investigate the relationship between the intensity of the bitterness with the contents of proteins and free amino acids. The results indicated that neither milk nor papaya alone tastes bitter. Heating the milk or the papaya before mixing and mixing only papaya latex with milk confirmed that an enzyme in papaya causes the bitter taste in papaya milk. The intensity of bitterness positively correlated with the contents of total soluble protein, free amino acids and the phenylalanine and tyrosine/tryptophan contents. Analyses using different papaya accessions in different seasons showed that tyrosine/tryptophan (r = 0.613***) and phenylalanine (r = 0.612***) correlate more strongly with bitterness intensity than the total soluble protein (r = 0.258*) or free amino acids (r = 0.38**). In this drink, milk provides the substrates to form the bitter substances, but the enzymes in the papaya are needed for the reaction to occur. The levels of the amino acids phenylalanine and tyrosine/tryptophan showed the highest correlation with the intensity of bitterness.