Rotating drum chambers are simple and effective devices for retaining particles in airborne state for prolonged periods. Many studies including inhalation toxicology, environmental fate, and survivability of airborne pathogens could benefit from using them. Particle size is the major factor governing aerosol suspension. Yet reliable experimental data as a function of particle size on the optimal rotation rate are limited. Therefore, this study aims to experimentally characterize a rotating drum and to optimize the operation rate for the bioaerosol size spectrum. Moreover, the sampling methodology for evaluating the performance of a rotating drum is also investigated. Charge-neutralized potassium sodium tartrate (PST) particles generated by an ultrasonic atomizer are used as surrogates of bioaerosols to characterize the performance of the rotating drum. Aerosol number concentrations and size distributions in the rotating drum are continuously or intermittently measured by an aerodynamic particle sizer (APS) in real time. Then the decay constants of aerosol number concentrations as a function of particle size, elapsed time, and rotation rate are calculated, respectively. Experimental results revealed that the rotation of a drum chamber enhances particle suspension greatly and the rotating rate can be optimized to retain the suspended particles for an extended time period. With the current drum geometry, the optimal rotation rate varies from 2 to 7 rpm in the particle size range of 1 to 7 µm and is proportional to particle size. When operating at the optimal rotation rate (2-4 rpm), 5% of 1-µm particles can remain suspended for over 24 hours. However, it is crucial to adjust the optimal rate carefully for large particles and long suspension duration.