Objective: The objective of this work is to prove and validate Tangential Flow Filtration System for high yield drug substance of vaccine as product and also to provide the experimental insight for vaccine industrial personnel to acquire the knowledge on the process validation of the system to obtain high and consistent yield of the product. Methods: The method in this work involves the experimental determination of the process parameters for Tangential Flow Filtration system such as Trans-membrane pressure and flux including validation of these parameters to achieve the high yield drug substance with consistency. The experiment entails the execution of three validation batches with different variables during the process and standardized at the point of maximum yield with similar reliability. Results: The performed validation of Tangential Flow Filtration system was so optimal and suitable for identification of parameters that controlled the yield of the drug substance consistently and validated accordingly. Statistical evaluation of the optimized parameters results from three consecutive batches was done and the mean was calculated individually as 0.25 bar for TMP, 0.4 LMH for flux and 402.6mg/mL for drug substance yield. Conclusion: The considered approach in the process validation of Tangential Flow Filtration system includes independent verification and application of parameters, which supports maximum yield of the vaccine product. The execution of these batches was successfully carried out in the laboratory that involves the purification of the upstream harvest sample for obtaining the maximum and consistent yield concentration of the drug substance with the decisive designing and validation of process parameters.