Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).