Pancreatic cancer is a devastating disease and early diagnosis and treatment are essential to improve the prognosis. We previously showed that α-fibrinogen containing hydroxylated proline residues at positions 530 and 565 is increased in plasma of pancreatic cancer patients. However, no antibody specific for hydroxylated proline-530 is available. Therefore, the purposes of this study were to develop a quantification method specific for both proline-hydroxylated α-fibrinogens by selected/multiple reaction monitoring (SRM/MRM), and to validate these modifications as pancreatic cancer markers. The target peptide for hydroxylated proline-530 contained methionine, and since variable partial oxidation of this residue would affect the quantification, hydrogen peroxide treatment was carried out to ensure complete oxidation. Quantification values of modified and unmodified α-fibrinogen were well correlated with those obtained by immunoblotting. Concentrations of modified and unmodified α-fibrinogen were quantified in 70 pancreatic cancer patients and 27 healthy controls. Percent hydroxylation of α-fibrinogen and concentration of hydroxylated α-fibrinogen were significantly greater in the plasma of patients. Furthermore, among 8 carbohydrate antigen 19-9 (CA19-9)-negative patients in stages I/II, 6 were positive for proline-hydroxylated α-fibrinogen. These results indicate that plasma concentration of proline-hydroxylated α-fibrinogen measured by SRM/MRM analysis may be a good pancreatic cancer marker, especially in CA19-9-negative patients.