The analytic process for identification of anaerobic bacteria involves direct specimen Gram stains and workup of growth in culture. Direct Gram stains help to develop the initial differential, aiding in guiding both the extent of laboratory workup and clinician decision making for patient management. Workup of anaerobic growth should involve an aerotolerance test to assess aerobic growth and to compare growth rates in anaerobic versus aerobic environments. While matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has become a routine means of anaerobe identification for many clinical laboratories, spot biochemical tests, such as indole and catalase, should be available in every laboratory that identifies anaerobes. If MALDI-TOF MS and biochemical methods are unsuccessful at providing an identification, 16S rRNA gene PCR and/or sequencing may be performed if the information impacts clinical care. The extent of culture workup to the level of presumptive identification versus definitive identification depends upon several factors, including the specimen source, direct Gram stain results, and the presence or absence of mixed microbiota. In addition to discussing the current best practices for anaerobic culture workup, commonly encountered anaerobic organism Gram stains and culture growth are detailed.