Available lysine content is an indicator of protein quality and nutritional value of milk. Many studies have examined the effects of extraction, treatment and storage of human milk upon its components, though no references are found regarding the possible changes in milk quality as defined by its content in essential amino acids such as lysine. The present study investigates the available lysine content in human milk and the variations in lysine resulting from milk manipulation as follows: (a) Cold storage (refrigeration at 4°C for 48 hours, and frozen for 15 days at −20°C); (b) Thermal treatment under conditions of low (Holder)(63°C/30 minutes) and high pasteurization (75°C/15 seconds). The results obtained show a decrease in milk lysine concentration after storage in both refrigerated and frozen samples. Pasteurization causes a highly significant loss of available lysine. The lysine losses were greater on applying low pasteurization versus the more gentle conditions of high pasteurization. Conclusions: While manipulation through cold storage or thermal treatment does not affect the protein content of human milk, its protein quality is modified. When human milk must be subjected to hygienization, it is preferable to apply high temperature treatment (75°C, 15 seconds) than habitual pasteurization (63°C, 30 minutes).