This study reports on the intestinal permeability of salt-containing mixtures of amino acids extracted from codfish salting wastewater. Permeability was evaluated in vitro using the Caco-2 cell line model; cell integrity during exposure to mixtures of amino acids was estimated by measuring the transepithelial electrical resistance (TEER). The effect of salt (NaCl) on the permeability and on the intestinal cell's integrity was also examined. Permeation rate (i.e. transport) was ≥95% for all amino acids except for creatine, for which it was 6%. Values for apparent permeability coefficients, Papp > 10−5 cm s−1, for mixture with isotonic concentration of NaCl suggest that amino acids are very likely to be absorbed in humans. Mixture with a hypertonic level of NaCl exerts a cytotoxic effect in intestinal cells resulting in a loss of epithelium integrity. Results show that isotonic mixture of amino acids extracted from codfish salting wastewater could be used in food, feed, cosmetic and pharmaceutical formulations. These applications could contribute to the fish industry sustainability.