The efficiency of respirators is usually determined by the protection factor, which is the ratio of the particle concentration outside the respirator to that inside the respirator. Most studies on workplace protection factors (WPF) of respirators have focused on the measurement of total mass concentrations, ignoring the effect of particle size. Furthermore, there appear to be no previous studies on protection factors against biological particles. In this study, a prototype personal sampling setup was developed for determining the protection provided by respirators against non-biological and biological particles in the size range of 0.7–10 μm. The range covers respirable and thoracic dust particles as well as most bacterial and fungal spores. The setup is compatible for field use in workplace environments and was optimized by minimizing particle losses in its aerosol transmission system. Theoretical modeling, laboratory tests, and field tests were performed for design optimization. After accounting for aerosol deposition mechanisms due to gravity, inertia, and turbulence affecting aerosol transmission through the straight and bending sections of specialized tubing, the theoretical data showed best agreement with the laboratory and field data for a tube diameter of ½ inch (~1.27 cm) among the three tested diameters. Tubing of this diameter also had the least amount of particle losses, and can be directed either above the ear or above the shoulder of the person whose respiratory protection is being evaluated. In addition, the ability of the setup to measure the WPF when a human subject donned a respirator was demonstrated successfully during soybean unloading. This study suggests that the new setup is a promising tool for future studies on evaluating respiratory protection against airborne dusts and microorganisms in occupational environments.