Elevated plasma levels of the biomarker soluble urokinase plasminogen activator receptor (suPAR) are prognostic of adverse outcomes in cardiovascular disease (CVD). This novel inflammatory protein has five known N-linked glycosylation sites, but the contribution of glycosylation to disease progression is unknown. This study aimed to assess the impact of glycosylation on suPAR measurements. Bio-banked plasma samples from healthy volunteers and patients with CVD were treated with deglycosylation enzymes. suPAR was measured for all treated and untreated samples using the Virogates suPARnostic ELISA. Percentage change in suPAR concentrations following treatment were compared using paired t-test, and differences between patient cohorts were examined using the Mann-Whitney test. In healthy subjects, suPAR concentrations increased after pre-treatment with deglycosylation enzymes (median 1.8 vs 2.4 ng/mL; p<0.001; n=15). In patients diagnosed with myocardial infarction (MI), a percentage increase in suPAR concentrations was also obtained after deglycosylation treatment, and this increment was higher than acute chest pain patients without MI (58% vs 44%; p=0.02; n=15). In patients with acute decompensated heart failure, a higher percentage change in suPAR concentrations was also observed following deglycosylation, compared with that of healthy subjects (38% vs 30%; p=0.04; n=33). suPAR immunoreactivity increased for the ViroGates ELISA after removal of protein glycosylation, as demonstrated by higher concentrations observed after treatment. This suggests that the current clinical assay for suPAR is influenced by its N-linked glycosylation. As glycosylation alterations are associated with CVD, further investigations are required to assess this implication with respect to the prognostic accuracy of suPAR in cardiovascular disease progression.