For decades the Great Lakes have been subject to invasive species introductions through the discharge of ships' ballast water. Several treatment technologies involving physical, chemical, and biological processes have been developed to remove or inactivate organisms in this discharge. Assessing the efficacy of these technologies involves estimating the number of viable propagules in treated discharge relative to untreated controls. For organisms in the 10–50 µm size range, for example, the International Maritime Organization (IMO) mandates that fewer than 10 viable organisms per milliliter may be discharged. To date, however, there is no standard method to assess viability of natural assemblages of organisms in this size group (largely phytoplankton and protozoans) in freshwater environments. We report here on a process of assemblage concentration, staining with fluorescein diacetate (FDA), and microscopic observation as a reliable and efficient method to assess densities of viable freshwater organisms in this size category in ballast discharge. A number of other methods, including digestion with enzymes, flow cytometry, and a variety of vital and mortal stains, were tested and discarded during this vetting process due to inconsistent or ambiguous results.