Rotating drum chambers are simple and effective devices for retaining particles in the airborne state for prolonged periods. Many studies, including inhalation toxicology, environmental fate, and survivability of airborne pathogens, can benefit from using them. Particle size is the major factor governing aerosol suspension, yet reliable experimental data on the optimal rotation rate as a function of particle size are limited. Therefore, this study aims to experimentally characterize a rotating drum and to optimize the rotation rate for the bioaerosol size spectrum. Moreover, the sampling methodology for evaluating the performance of a rotating drum is 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. The aerosol number concentrations and size distributions in the rotating drum are both continuously and intermittently measured by an aerodynamic particle sizer (APS) in real time. Then, the decay constants of the aerosol number concentrations as functions of particle size, elapsed time, and rotation rate are calculated. The experimental results reveal that the rotation of the drum chamber greatly enhances particle suspension and the rotation rate can be optimized to prolong the suspension for an extended period. With the current drum geometry, the optimal rotation rate varies from 2 to 7 rpm for the particle size range of 1 to 7 µm and is proportional to the particle size. At the optimal rotation rate (2–4 rpm) for 1-µm particles, 5% of them can remain suspended for over 24 hours. However, this rate must be adjusted for large particles in order to maximize the period of suspension.