Most of the experiments oriented to the nutritional evaluation of different feed ingredients for aquacultured organisms are focused to the determination of digestibility coefficients for the main nutrients particularly those of the protein fraction. Nevertheless, fish nutritionists are well aware of the difficulties and errors obtained when digestibility measurements are carried out in vivo, due to the special features of the aquatic environment and water-living organisms. At present, digestibility trials performed in vivo may be complemented, and in some cases preceded or substituted, by several biochemical techniques offering a wide range of practical applications. A three-step approach is proposed as a good way for obtaining valuable results when designing in vitro digestibility trials. Such an approach should begins with the appraisal of specific activities of selected digestive enzymes, followed by the characterisation of such activities, and finally by the utilization of this information in order to simulate the digestive process taking place in fish. Possibilities and limitations of in vitro techniques operating in either closed or open systems are discussed.