The objectives of this study were: (a) to evaluate the effect of inclusion of mechanically separated chicken meat (MSCM) in dry dog food on fatty acid profile, in vivo and in vitro digestibility, and palatability as compared with dry dog food containing meat by-products (MBP); (b) to determine, whether or not, the inclusion of the one or the other ingredient changes the microbiology and the storage quality of the two food products; (c) to propose a new system (DaisyII Incubator) to measure the in vitro digestibility of the two products. Their similar chemical composition notwithstanding, the MSCM product had lower palatability but better nutritional quality (with higher polyunsaturated fatty acid [PUFA] content and lower saturated fatty acid [SFA] content) than the MBP product. Microbiological risk assessment showed no microbiological hazards for either product. After 6 months storage, polyamine values were found to be higher in the MSCM than in the MBP. Finally, the DaisyII Incubator proved a valid instrument for the study of in vitro digestibility also for dogs; since it provided data simply, quickly with less variability and cost than obtained with in vivo trials, it could represent the future for pet food digestibility studies. Our results indicate that inclusion of MSCM or MBP as the main protein ingredient in extruded pet food may be used advantageously in product formulations.