The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.