This protocol describes a procedure for quantitative determination of microbial contamination in a nanoparticle preparation. Nanoparticle samples, along with controls, are spread on the surface of agar and growth of bacterial colonies is monitored after 72 hr of incubation. The intended purpose of this assay is to avoid introduction of microbial contamination into in vitro cell cultures and in vivo animal studies utilizing the test-nanomaterial, as microbial contamination will confound the results of these tests. This assay is not intended to certify the material as sterile.