A one-step sandwich chemiluminescence immunometric assay (LIA) was developed for the quantification of bifunctional peptidylglycine-α-amidating monooxygenase (PAM) in human plasma (PAM-LIA). PAM is responsible for the activation of more than half of known peptide hormones through C-terminal α-amidation. The assay employed antibodies targeting specific catalytic PAM-subunits, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), to ensure detection of full-length PAM. The PAM-LIA assay was calibrated with a human recombinant PAM enzyme and achieved a detection limit of 189 pg/mL and a quantification limit of 250 pg/mL. The assay demonstrated good inter-assay (6.7%) and intra-assay (2.2%) variabilities. It exhibited linearity when accessed by gradual dilution or random mixing of plasma samples. The accuracy of the PAM-LIA was determined to be 94.7% through spiking recovery experiments, and the signal recovery after substance interference was 94–96%. The analyte showed 96% stability after six freeze–thaw cycles. The assay showed strong correlation with matched EDTA and serum samples, as well as matched EDTA and Li-Heparin samples. Additionally, a high correlation was observed between α-amidating activity and PAM-LIA. Finally, the PAM-LIA assay was successfully applied to a sub-cohort of a Swedish population-based study, comprising 4850 individuals, confirming its suitability for routine high throughput screening.