Detection of antineutrophil cytoplasmic antibodies (ANCAs) is a well-established diagnostic test used to evaluate suspected necrotizing vasculitis of small blood vessels. Conditions associated with these antibodies, collectively referred to as ANCA-associated vasculitides, include granulomatosis with polyangiitis (formerly known as Wegener granulomatosis), microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis (formerly known as Churg-Strauss syndrome). The diagnostic utility of ANCA testing depends on the type of assay performed and on the clinical setting. Most laboratories worldwide use standard indirect immunofluorescence tests (IFT) to screen for ANCA and then confirm positive IFT results with antigen-specific tests for proteinase 3 (PR3) and myeloperoxidase (MPO). Developments such as automated image analysis of immunofluorescence patterns, so-called third-generation PR3-ANCA and MPO-ANCA ELISA, and multiplex technology have improved the detection of ANCAs. However, challenges in routine clinical practice remain, including methodological aspects of IFT performance, the diverse antigen-specific assays available, the diagnostic value of testing in clinical settings and the prognostic value of serial ANCA monitoring in the prediction of disease relapse. This Review summarizes the available data on ANCA testing, discusses the usefulness of the various ANCA assays and advises on the clinical indications for the use of ANCA testing.