This study compared the brain proteome response of fish submitted to cold shock by fast-chilling (FC) as a novel stunning method (with fish immersed in liquid water at -8ºC), asphyxia (ASP) (as the currently used method), electrical stunning (ES) and anaesthesia (AN) with MS-222, by integrating it with physiological, behavioural and quality welfare indicators. Fish trials were conducted in triplicate tanks and two fish per tank were randomly sampled for MS analysis. One of the objectives of this integrative work was to disclose most significant changes in the brain proteome of rainbow trout (O. mykiss) subjected to four types of stunning methods that could help to elucidate about welfare standards.