Background: Indirect immunofluorescence (IIF) using HEp-2 cells is internationally recognized as the gold-standard method for detecting antinuclear antibodies (ANAs), which are widely used in the diagnosis of systemic autoimmune diseases. Developing and evaluating a standardized experimental protocol for ANA detection is important for improving the diagnosis of systemic autoimmune diseases. Aim: To detect serum ANAs using an HEp-2 cell-based indirect immunofluorescence assay. Materials and Methods: Frozen HEp-2 cells were thawed, recovered, and cultured. Cells were seeded at a density of 5x10⁵ cells per reaction well and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 hours, followed by fixation. The fixed cells were incubated with serum samples from patients diagnosed with systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SjS), diluted at 1:80 and 1:160. A fluorescein isothiocyanate (FITC)-conjugated secondary antibody was subsequently applied at a dilution of 1:500. Fluorescence patterns were examined using an immunofluorescence microscope at 20× and 40× magnifications. Nuclear staining patterns observed at a serum dilution of 1:160 under 40× magnification were evaluated with reference to the International Consensus on ANA Patterns (ICAP) classification. Results: HEp-2 cells were evaluated using the indirect immunofluorescence (IIF) assay with ANA-positive and ANA-negative sera. No fluorescence was observed in the negative control serum. In contrast, serum positive for anti-dsDNA from a patient with systemic lupus erythematosus (SLE) produced a distinct homogeneous nuclear staining pattern, whereas anti-SSA/SSB-positive serum from a patient with Sjögren’s syndrome (SjS) produced a speckled nuclear staining pattern. The homogeneous and speckled staining patterns observed in the respective SLE and SjS sera were consistent with the expected morphological characteristics of the corresponding reference patterns, supporting the applicability of the established HEp-2 cell-based IIF protocol for ANA detection and nuclear staining pattern assessment. Conclusion The HEp-2 cell-based indirect immunofluorescence (IIF) assay enabled the detection of serum ANAs and the differentiation of homogeneous and speckled nuclear staining patterns. The observed staining patterns were morphologically consistent with the corresponding reference patterns described in the International Consensus on ANA Patterns (ICAP) classification. These findings support the potential applicability of the established protocol for ANA detection and nuclear staining pattern assessment.