Nano/micro polystyrene (PS) beads have found many applications in different fields spanning from drug delivery, bio-diagnostics, and hybrid plasmonics to advanced photonics. The sizes of the PS beads are an important parameter, especially in plasmonic and photonic experiments. In this work, we demonstrate a quick and straightforward method to estimate the diameters of sub-microspheres (0.2 μm to 0.8 μm) using the transmission spectra of a close-packed monolayer of polystyrene beads on glass or quartz substrates. Experimental transmission spectra of the PS monolayers were verified against finite-difference time-domain (FDTD) simulation and showed good agreement. The effects of the substrates on the transmission spectra and, hence, the accuracy of the method were also studied by simulation, which showed that common transparent substrates only cause minor deviation of the PS bead sizes calculated by the proposed method.