Azospirillum is a genus that possesses 37 species, and only one does not fix nitrogen. A. brasilense and A. lipoferum were the two species described and the former has been used in agriculture in many countries since its identification in 1978. A. argentinense is an inoculant recommended in Argentina for more than 30 years. These species are well-known bacteria that produce high amounts of auxins, affecting the root architecture by its inoculation and the visual symptoms associated with their application on plants. The main strategy used to isolate and count this bacterial genus is based on the utilization of semisolid medium without nitrogen addition. This recipe has been used by many authors since 1976. This chapter presents procedures used for diazotrophic counting and identification either from rhizosphere soil or on the surface of or within plant tissues. It also includes methods to measure nitrogenase activity in vials with the semisolid medium, carbon source characterization, and storage procedure. We also included analyses and discussion of techniques based on phylogeny, phylogenomics, average nucleotide identity (ANI), and mass spectrometry using type strains of Azospirillum species to guide the correct characterization of species in this genus.