Background Due to the huge amounts of their production in Europe, their environmental impact, and the difficulty in processing them, there is a clear necessity for the valorization of rainbow trout viscera. Considering that the production of fishmeal with viscera can be problematic, and in order to make viscera more profitable, the production of fish protein hydrolysates has been considered. Although silage and enzymatic hydrolysis are the most common methods for obtaining hydrolysates, autolysis has emerged as an alternative method that uses endogenous enzymes of the viscera. Methods Considering the stability and characteristics of the enzymes, a factorial design was carried out using three variables: pH, temperature, and water content. The design resulted in 15 experiments, and the results were analyzed using response surface methodology. The optimum parameters were validated by comparing the predicted outcomes with experimental results. Additionally, a kinetics study was conducted to shorten the autolysis time. Results from autolysis were compared with those from silage and enzymatic hydrolysis in a previous study. Results The optimal conditions for achieving the highest degree of hydrolysis and yield of free amino acids (FAAs) per 100 g of viscera and per total protein were determined to be a pH of 8, a temperature of 40 ºC, and a water content of 6.85%. The pH and content of the added water were found to be significant variables during autolysis (p < 0.05). The kinetic study showed that 7 h was still required to be effective. Conclusions Autolysis achieved a lower degree of hydrolysis than silage; however, as it solubilized more protein, the global yield of free amino acids per 100 g of viscera was slightly higher. It was concluded that endogenous alkaline proteases could be used in an autolytic process to obtain a free amino acid-rich hydrolysate from trout viscera.