The optimum hydrolysis time for simulative digestion stages was investigated to provide a reference for designing the process of in vitro digestion in layer feedstuff. A single-factor randomized design was adopted to study the gastric and intestinal simulative digestion time. There were 6 treatments (1, 2, 3, 4, 5 and 6 h) and 3 treatments (12, 15 and 18 h) of digestion time in gastric and intestinal in vitro digestion stage, respectively. Each treatment contained 5 replicates with 1 simulative digestive tube per replicate. The dry matter digestibility and enzymatic hydrolysate energy value in different time were measured. The results showed as follows: in gastric simulative digestion stage, there were not significant differences in dry matter digestibility and enzymatic hydrolysate energy value of corn-soybean meal diet and corn-soybean meal-cottonseed meal diet digested for 4, 5 and 6 h （P>0.05）, which were significantly higher than those measured at 1, 2 and 3 h (P<0.05). In intestinal simulative digestion stage, the differences of 12, 15 and 18 h dry matter digestibility of corn-soybean meal diet were not significant (P>0.05), but the 12 h enzymatic hydrolysate energy value was significantly lower than that of 15 and 18 h (P>0.05). The dry matter digestibility and enzymatic hydrolysate energy value of corn-soybean meal-cottonseed meal diet were significantly increased from 12 to 15 h (P<0.05), but no longer increased after being digested from 15 to 18 h. In conclusion, the optimum time of gastric and intestinal simulative digestion is 4 and 15 h, respectively.［Chinese Journal of Animal Nutrition, 2010，22（5）:1422-1427］