ObjectivesTo validate a rapid serological test (RST) for SARS-CoV-2 antibodies used in seroprevalence studies in healthcare providers, including primary healthcare providers (PHCPs) in Belgium.DesignA phase III validation study of the RST (OrientGene) within a prospective cohort study.SettingPrimary care in Belgium.ParticipantsAny general practitioner (GP) working in primary care in Belgium and any other PHCP from the same GP practice who physically manages patients were eligible in the seroprevalence study. For the validation study, all participants who tested positive (376) on the RST at the first testing timepoint (T1) and a random sample of those who tested negative (790) and unclear (24) were included.InterventionAt T2, 4 weeks later, PHCPs performed the RST with fingerprick blood (index test) immediately after providing a serum sample to be analysed for the presence of SARS-CoV-2 immunoglobulin G antibodies using a two-out-of-three assay (reference test).Primary and secondary outcome measuresThe RST accuracy was estimated using inverse probability weighting to correct for missing reference test data, and considering unclear RST results as negative for the sensitivity and positive for the specificity. Using these conservative estimates, the true seroprevalence was estimated both for T2 and RST-based prevalence values found in a cohort study with PHCPs in Belgium.Results1073 paired tests (403 positive on the reference test) were included. A sensitivity of 73% (a specificity of 92%) was found considering unclear RST results as negative (positive). For an RST-based prevalence at T1 (13.9), T2 (24.9) and T7 (70.21), the true prevalence was estimated to be 9.1%, 25.9% and 95.7%, respectively.ConclusionThe RST sensitivity (73%) and specificity (92%) make an RST-based seroprevalence below (above) 23% overestimate (underestimate) the true seroprevalence.Trial registration numberNCT04779424.