Abstract: OBJECTIVE to select perfect lactose concentration instead of TSB as the liquid culture medium to execute simulate filling at the liquid section which can reach the final API aseptic filling simulation requirements. METHODS Using the characteristics of lactose and the established final sterilization workshop of sterile API manufacturing process, investigated the growth promotional ability of different concentration of lactose, and then selected the appropriate lactose concentration for methodology validation. Finally this lactose was used instead of TSB culture medium to carry out filling simulation validation. RESULTS Using 5% and 7% lactose could be the simulate filling and the lower concentration of 5% lactose was recommend for its easier cleaning. CONCLUSION The 5% lactose instead of 3% TSB as the liquid culture medium to execute simulate filling at the liquid section can reach the final API aseptic filling simulation requirements.