An electrospray mass spectrometry-based methodology has been developed to have a fast and sensitive method for protein-cofactor stoichiometry determination. As model systems, we used two proteins which require the presence of cofactors for activity: TraR, a member of the LuxR family of quorum-sensing transcriptional regulators, which requires an acyl-homoserine lactone molecule called Agrobacterium autoinducer (AAI) as coinducer and the NS3 protease of hepatitis C virus which complexes with a NS4A cofactor peptide. Both TraR/AAI and NS3/NS4A are noncovalent complexes. Our method requires only nanomolar concentration of sample. A calibration curve of the cofactor is determined by high-performance liquid chromatography (HPLC) coupled on-line with an ion trap mass spectrometer operated in selected reaction monitoring mode. Subsequently, the complex is analyzed using the same experimental setup. During the HPLC run, the complex dissociates, and cofactor and protein elute at different retention times. The peak area of the cofactor is integrated and the molar concentration of cofactor in the complex is extrapolated from the calibration curve. The stoichiometry is consequently calculated by dividing the molar concentration of protein injected by that of cofactor measured. Both TraR/AAI and NS3/NS4A complexes have 1:1 stoichiometries, in line with those already reported in the literature.