This protocol describes a procedure for detection and 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. Additionally, to detect autotrophic and oligotrophic bacteria which grow in media containing low nutrient levels and may contaminate cell culture after passing rich medium agar plate test, the same nanoparticle samples are spiked into RPMI and incubated up to 7 days. 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. The second goal of this test is to determine bacterial identity in samples found to be positive. The ID results can be helpful in determining the source of contamination. This assay is not intended to certify the nanomaterial as sterile.