The egg white and egg yolk are the two main sources of nutrients for the developing avian embryo. Egg white ultimately reaches egg yolk before being consumed by the embryo. Previously, in the turkey Meleagris gallopavo, we showed that the major egg white transfer into the yolk through the amnion and intestinal lumen of the embryo started on day 17 and became obvious on day 19. We also suggested that the transferred egg white and endogenous yolk proteins undergo digestion at late developmental stages. However, the protease activity throughout development and the type of activated proteases are completely unexplored in the yolk of turkey eggs. Here, we measured the general proteolytic activity in the egg yolk during different developmental stages using casein as a substrate. Furthermore, we determined the type of activated proteases by employing different types of protease inhibitors. Protease activity in the egg yolk was basal throughout development until day 19 when activity significantly increased, remaining high thereafter. Moreover, acidic aspartic protease(s) were primarily activated in the yolk. Our results suggest that the transferred egg white and endogenous yolk proteins are mostly degraded by the aspartic proteases that become highly activated late in development.