Antiprothrombin antibodies, measured with phosphatidylserine/prothrombin complex (aPS/PT) ELISA, have been reported to be associated with antiphospholipid syndrome (APS). They are currently being evaluated as a potential classification criterion for this autoimmune disease, characterized by thromboses and obstetric complications. Given the present lack of clinically useful tests for the accurate diagnosis of APS, we aimed to evaluate in-house and commercial assays for determination of aPS/PT as a potential serological marker for APS. We screened 156 patients with systemic autoimmune diseases for antibodies against PS/PT, β₂-glycoprotein I, cardiolipin and for lupus anticoagulant activity. We demonstrated a high degree of concordance between the concentrations of aPS/PT measured with the in-house and commercial assays. Both assays performed comparably relating to the clinical manifestations of APS, such as arterial and venous thromboses and obstetric complications. IgG aPS/PT represented the strongest independent risk factor for the presence of obstetric complications, among all tested aPL. Both IgG and IgM aPS/PT were associated with venous thrombosis, but not with arterial thrombosis. Most importantly, the association between the presence of IgG/IgM aPS/PT and lupus anticoagulant activity was highly significant. Taken together, aPS/PT antibodies detected with the in-house or commercial ELISA represent a promising serological marker for APS and its subsets.