Abstract In the present study, HPLC–ESI–IT (ion-trap) MS was used for carboxyterminal (C-terminal) amino acid sequence confirmation of intact recombinant Hirudin Variant 3 (HV3) and alkylated HV3. The C-terminal amino acid sequence of HV3 was determined by the use of carboxypeptidase P (CPP), and by the combined use of carboxypeptidase P and carboxypeptidase Y (CPY). The C-terminal amino acid sequence of alkylated HV3 with 1,4-dithiothreitol (DTT) reduction was also confirmed by the combined use of CPP and CPY (abbreviated to CPP-CPY). Up to 19 amino acid residues were confirmed in the nanomolar concentration range by analyzing the molecular weights of the truncated peptides of HV3. Another five amino acids were confirmed in the nanomolar concentration range of alkylated HV3 with DTT reduction. For sequencing alkylated HV3 with DTT reduction, HV3 reduced with DTT followed by alkylation with iodoacetamide. The reaction mixture, which included alkylated HV3, DTT, and iodoacetamide, was then directly sequenced without any further pre-treatment. The reaction was designed in a time-, and concentration-dependent manner to obtain the maximum sequence information. The results showed that HPLC–ESI–ITMS cannot only determine the C-terminal amino acid sequence of HV3, but also gives important information about the enzymatic degradation and subsequent release of the C-terminal amino acids of HV3.