Cleanrooms play an important role in various industries, such as pharmaceuticals, specifically for vaccine cleanrooms by strictly regulating contaminants, temperature, humidity, and pressure to ensure product reliability, particularly in critical areas. This study investigates a biotechnology vaccine cleanroom using a comprehensive field measurement test and numerical simulation analysis. Field measurements were conducted during the at-rest and operational occupancy states. As a result, it was discovered that the operational condition has a higher concentration than the at-rest condition. To mitigate this issue, the numerical simulation improvement strategy employed in this study aimed to reduce the concentration under operational conditions. The strategy proposed in this simulation is determining the ventilation rate and arranging the outlet air grilles. To assess and evaluate the performance of the cleanroom, we utilized ventilation and removal efficiency metrics. These indices served as important metrics for evaluating the cleanroom's efficiency in controlling contaminants. In at-rest conditions, ventilation and removal efficiency were 90.0 % and 86.2 %, respectively. However, under operational conditions, these metrics dropped to 81.3 % and 80.1 %, respectively, in the baseline case. After the proposed improvement design, the ventilation and removal efficiency in the operational condition could improve by up to 6.8 % and 5.2 %, respectively. A proper arrangement of outlet air grilles could control the decrease in concentration. In addition, a higher ventilation rate also could decrease the concentration. However, it caused high energy consumption. Therefore, the ventilation rate must be designed optimally to compromise contamination control and energy saving.