Chronic kidney disease (CKD) patients suffer from a multitude of comorbidities, with vascular calcification being a major one. Recently, a new 19 amino acid long peptide, called calcification blocking factor (CBF), inhibiting vascular calcification has been identified. CBF concentration has been measured in a small cohort of patients by a mass-spectrometric work- and time-intensive approach, revealing reduced levels in patients with CKD stage 5. However, a reliable and time-efficient method for quantifying CBF concentration in large patient populations is still lacking. Developing a scalable and rapid assay is therefore essential to fully explore the clinical relevance of CBF in CKD progression. Therefore, we established a monoclonal antibody targeting the active domain of CBF using the hybridoma technique. Hybridoma clones were screened for antibody-quantity, -epitope recognition, and -specificity over a series of multiple ELISA approaches. Ultimately, one final clone emerged and was used to establish a monoclonal cell line, which was subsequently utilised for antibody production and purification. The purified antibody was characterised by surface plasmon resonance, indirect, and competitive ELISA. The competitive ELISA was used to measure plasma samples of three patient cohorts, including early and late-stage CKD patients, as well as non-CKD patients with mild to moderate coronary artery calcification. The results showed that not only patients with decreased renal function, but also those with calcified aortas, have a reduced CBF plasma concentration. We developed a new monoclonal antibody targeting the active domain of CBF, enabling high-throughput measurement of plasma samples. The developed antibody and the established ELISA provide a new approach to further analyse and understand the mechanisms underlying vascular calcification with focus on CBF.