Objectives: We aimed to assess and compare Anti-Cell (AC) staining patterns and semi-quantitative titre levels of related antibodies in patients with juvenile idiopathic arthritis (JIA) with and without associated uveitis (JIA-U) to identify distinctive immunological profiles and contribute to a deeper understanding of JIA-U aetiopathogenesis. Methods: Data from JIA patients diagnosed between January 2020 and December 2025 in our centre were evaluated. The HEp-2 indirect immunofluorescence assay (HEp-2 IFA) was used to determine AC patterns and semiquantitative titres of associated antibodies. Results: 217 JIA patients were evaluated: 161 (74%) without uveitis and 56 (26%) with JIA-U. The AC-1 pattern was detected in 94 of 217 children (43%). It was markedly enriched among children with JIA-U, present in 39 of 56 (70%), compared with 55 of 161 (34%) in the non-uveitis group. High staining intensity (i.e., semiquantitative titre ++/+++) of AC-1 was also more commonly detected in JIA-U patients (39%) compared to children without uveitis (11.8%). The AC-4 pattern was detected in 30 of 217 JIA patients (14%), but more commonly in the non-uveitis (17.4%) compared to the JIA-U group (3.6%). Conclusions: JIA-U is closely linked to AC-1 staining patterns and higher semiquantitative titres of associated antibodies. Our results provide additional insight into the pathogenesis of JIA-U and underscore the significance of the new Paediatric Rheumatology International Trials Organisation (PRINTO) JIA classification criteria. The potential clinical relevance of AC patterns in children with JIA and JIA-U requires further multicentre studies involving larger cohorts and extended follow-up periods.