The simplified criteria for the diagnosis of autoimmune hepatitis (AIH) include immunofluorescence testing (IFT) of antinuclear and smooth muscle autoantibodies (ANA and SMA) on rodent tissue sections. We aimed to establish scoring criteria for implementation of ANA IFT on HEp-2 cells and ELISA-based testing.ANA and SMA reactivity of 61 AIH sera and 72 non-alcoholic fatty liver disease (NAFLD) controls were separately assessed on tissue sections and human epithelioma (HEp-2) cells to compare the diagnostic value at increasing titers. A total of 113 AIH patients at diagnosis and 202 controls from three European centers were assessed by IFT as well as three different commercially available ANA ELISA and one anti-F-actin ELISA.ANA assessment by IFT on liver sections had 83.6% sensitivity and 69.4% specificity for AIH at a titer of 1:40. On HEp-2 cells, sensitivity and specificity were 75.4% and 73.6%, respectively, at an adjusted titer of 1:160. Area under the curve (AUC) values of ANA ELISA ranged from 0.70 - 0.87, with ELISA coated with HEp-2 extracts in addition to selected antigens performing significantly better. SMA assessment by IFT had the highest specificity for the SMA-VG/T pattern and anti-MF reactivity on HEp-2 cells. ELISA-based anti-F-actin evaluation was a strong predictor of AIH (AUC 0.88) and performed better than SMA assessment by IFT (AUC 0.77 - 0.87).At adjusted cutoffs, both ANA IFT using HEp-2 cells and ELISA-based autoantibody evaluation for ANA and SMA are potential alternatives to tissue-based IFT for the diagnosis of AIH.