Background: The prevalence of SARS-CoV-2 variants of concern (VOCs) has changed unevenly over time around the world. Although whole genome sequencing is the gold standard for virus characterization, the discovery of alpha VOC causing spike gene target failure (SGTF) result, when tested using an RT-qPCR assay, has provided a simple tool for tracking the frequencies of variants. Objectives: The aim of this study was to investigate if a multiplex RT-qPCR assay (BioM 4Plex VOC) could be used to detect SARS-CoV-2 and to perform a VOC screening test in a single reaction tube. Here, we present the multicenter study evaluating this assay. Methods: Twelve laboratories have participated in the multicenter study. The BioM 4Plex VOC was distributed to them with detailed instructions of how to perform the test. They were asked to test the BioM 4Plex VOC in parallel with their routine Commercial SARS-CoV-2 diagnostic assay. Additionally, they were requested to select SARS-CoV-2-positive samples with genome sequenced and lineage definition according to PANGO lineage classification. Findings: The BioM 4Plex VOC and commercial RT-PCR assay are equally effective in detecting SARS-CoV-2. Results revealed a specificity of 96.5 – 100% [95% confidence interval (CI)], a sensitivity of 99.8 – 100% (95% CI), and an accuracy of 99.8 – 100% (95% CI). A 99% concordance rate was found between results from the BioM 4Plex VOC and that from available genome sequencing data. Main conclusions: The BioM 4Plex VOC provides an effective solution to detect SARS-CoV-2 infections and screening for VOCs in a single reaction. It is a straightforward method to help us monitor the frequency and distribution of VOCs and develop strategies to better cope with the pandemics.