Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria (anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III). However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP) that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9) are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies. A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n = 32) and Italy (Careggi Hospital, Florence, n = 15) selected based on AC-9 HEp-2 IFA staining (> 1:640, clumpy nucleolar pattern) and 102 non-SSc controls (inflammatory bowel disease (IBD) n = 20, Sjögren's syndrome (SjS) n = 20, infectious disease (ID) n = 7, systemic lupus erythematosus (SLE) n = 17, rheumatoid arthritis (RA) n = 17, and healthy individuals (HI) n = 21). All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples. The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0%, Italy) of samples preselected based on the AC-9 IIF pattern (difference p = 0.09). Collectively, the PMAT assay showed 91.5% (95% confidence interval (CI): 80.1-96.6%) sensitivity with 100.0% (95% CI: 96.4-100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman's rho = 0.89, 95% CI: 0.77.9-0.95%, p < 0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ). In conclusion, this first study on anti-fibrillarin antibodies measured using a novel PMAT assay shows promising results where the new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. The availability of novel AFA assays such as PMAT might facilitate the clinical deployment, additional studies, standardization efforts, and potentially consideration of AFA for next generations of the classification criteria.