Anti-mitochondrial autoantibodies (AMA) are specific markers of primary biliary liver cirrhosis (PBC), a cholestatic autoimmune disease which is characterised by a progressive destruction of the biliary epithelial cells followed by fibrosis, cirrhosis and liver failure. The prevalence of AMA in PBC is more than 90% and they can precede long before the clinical symptoms. AMA are conventionally detected by indirect immunofluorescence (IIF) using rodent liver, kidney, and stomach sections as substrates. Additionally, different PBC-specific anti-nuclear autoantibodies (ANA) can be observed in 30% of patients presenting with multiple nuclear dot or nuclear membrane staining patterns, which preferentially are identified using HEp-2 cells as substrate. The identification of the major PBC-specific mitochondrial and nuclear targets has allowed the generation of monospecific antigenic targets which are increasingly used in solid-phase assays for routine detection of AMA and ANA in mono- or multiparametric screen test systems. In the present paper, we give an overview of the diagnostic significance of autoantibodies in PBC, discuss the competencies of different techniques used for their determination and propose an effective diagnostic strategy.