We evaluated a method for the assessment of bacterial viability that is based on ultraviolet and visible (UV-Vis) spectroscopy analysis. The quantities of intracellular materials inside a cell vary depending on change of bacterial viability by disruption of the membrane integrity. Therefore, normalized optical density in the range of 200–290 nm was analyzed to determine if it varied in samples containing different proportions of live bacteria. Our results indicate that samples containing higher proportions of live bacteria such as Escherichia coli, Bacillus subtilis, and Staphylococcus epidermidis had higher optical densities. In addition, the optical density at 230 nm divided by the optical density at 670 nm was found to have a strong linear correlation with bacterial viability (the R2 values of E. coli, B. subtilis, and S. epidermidis are 0.9964, 0.9118, and 0.9861, respectively). Our proposed rapid assessment method takes less than three minutes and only requires optical measurements at 230 and 670 nm; therefore, it is simpler and faster than colony counting, fluorochromasia, or the dye-exclusion test. Moreover, our method was applied to bioaerosols, which are currently important issues in public health, microbiology, aerosol science, and other fields. In our study, the bacteria (E. coli) were dispersed into the air using a Collison-type atomizer, and were sampled in sterilized deionized water using an impinger with a pump. According to our method, the viability of E. coli was approximately 55.2%, which was similar to 52.5 ± 4.7% determined from the LIVE/DEAD BacLight bacterial viability assay.