To evaluate the effect of non-animal protein hydrolysates on keratinocyte differentiation, commercially-available soy protein hydrolysates were employed as feed supplements for serum-free culture medium in the absence or presence of bovine pituitary extract (BPE). Supplementation of keratinocyte culture medium containing BPE with Soy Hydrolysate (1 g/L) or Bacto Soytone (1 or 2 g/L) inhibited the mRNA and protein expression levels of early and late differentiation markers, and also helped cells to maintain a small size, an indication of an undifferentiated state. These results show that the supplementation of protein hydrolysates might be helpful to delay keratinocytes differentiation.