Different from the rigid separation of biological tissue by scalpel, medical waterjet technology utilizes the impact kinetic energy of high-speed waterjet to instantly destroy the surface of biological tissue to achieve better clinical separation effect. Since the gelatin samples are transparent biomaterials, they were taken as the only substitute for soft tissue in experimental studies and observation in the current studies of medical waterjet separation technology, but the mechanical behavioural difference between the two has not been reported. To verify the adaptability of gelatin as a substitute of soft tissue under the impact of high-speed waterjet. Firstly, the dynamic process of impact was described through the energy balance equation. Then, based on the principle of altering a single variable, the difference of damage depth between 8 wt. %, 10 wt. %, and 12 wt. % gelatin samples and porcine liver tissue under various impact pressure, impact distance, and waterjet speed of motion (as opposed to flow velocity) was compared. The results show that the gelatin sample replicates the damage behaviour of porcine liver samples under the specific waterjet impact conditions, and the direction of the waterjet hydraulic power optimisation of the two materials is also consistent: however, due to the different sampling sites and anisotropy of porcine liver samples, the mechanical response of soft tissue under high-speed waterjet impact cannot be fully expressed by gelatin samples. The experimental results can provide support for the further study of medical waterjet separation technology.INDEX TERMS High-speed impact, medical waterjet, separation behavior, surgical instruments, tissue engineering.