The aim of this study was to develop the first quantitative serological test for anti-SARS-CoV-2 antibodies in human serum with liquid chromatography - quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Other assays, mostly immunoassays, are only qualitative or semi-quantitative, and hence, actual antibody concentrations after SARS-CoV-2 infection are unknown. In our assay, anti-SARS-CoV-2 antibodies were isolated with spike protein subunit 1 (S1) coupled to magnetic beads. IgG1 signature peptide GPSVFPLAPSSK was selected for quantification using ipilimumab calibration standards and SILuMAb K1 as the stable-isotope labeled internal standard. The anti-SARS-CoV-2 IgG1 calibration range was from 1.35 to 135 nM. Inter-assay accuracies were between 98.8%− 107% with inter-assay precisions between 8.37%− 13.5% measured at 3 concentration levels on three separate occasions. Anti-SARS-CoV-2 IgG1 antibodies were quantified in PCR-positive patients with mild to severe symptoms. IgM signature peptide DGFFGVPR was detected in patients that recently recovered from COVID-19. A unique and quantitative LC-QTOF-MS method to quantify anti-SARS-CoV-2 IgG1 in serum was successfully developed and its clinical applicability has been demonstrated.