This study was conducted to test the laboratory reproducibility of digestion condition and determination result in simulated digestion system (SDS-2) for monogastric animals. A single factor completely randomized design was adopted with 4 laboratory treatments. Corn and soybean meal were ground then passed a 60-mesh and delivered to the 4 laboratories. In each laboratory, one SDS-2 was selected to measure the flow velocities of buffer solution, digestive fluid and washing solution. Variation curves of the temperature in enzymatic reaction room, buffer solution controlled room and digestive fluid storage room were measured by temperature sensors, variation curves of the mixing frequency was measured by electromagnetic sensor. The enzymatic hydrolysate gross energy (EHGE) of corn and soybean meal was determined according to the manual of SDS-2. The results showed as follows:1) there were significant differences in the flow velocity of digestive fluid in small intestine and large intestine among different laboratories (P<0.05), however, no significant differences were observed in the flow velocities of buffer solution in stomach, small intestine and large intestine and washing solution among different laboratories (P>0.05). There were significant differences in the flow velocities of buffer solution in stomach, small intestine and large intestine and washing solution in 2 simulated digestion groups in the same SDS-2 (P<0.05). 2) In the 4 laboratories, despite different initial mixing frequencies and initial temperatures in enzymatic reaction room, buffer solution controlled room, digestive fluid storage room, the consistent digestion condition was observed after the SDS-2 performed for 60 min. 3) In the corn, the intra-laboratory, inter-laboratory and total coefficient of variation (CV) were all less than 1.23% for dry matter digestibility (DMD) and EHGE, and no difference was observed across the laboratories (P>0.05). In the soybean meal, the intra-laboratory, inter-laboratory and total CV were 1.30%, 2.23% and 2.52% for DMD and 0.89%, 1.43% and 1.64% for EHGE, and significant difference was observed across the laboratories (P<0.01). In conclusion, the difference in the flow velocity of small intestinal fluid and large intestinal fluid does not lead to the difference in EHGE determination result across the laboratories. Among 4 laboratories, the consistent digestion condition can be attained after the SDS-2 performed for 60 min. The reproducibility of EHGE in corn across laboratories is better than that in soybean meal. The total CV of EHGE of 2 feedstuffs across laboratories is less than 1.64%, indicating the reproducibility is satisfactory.