SARS-CoV-2 clinical samples can be detected as positive for a long period of time using real-time RT-PCR, even when patients are no longer infectious. Viral culture is the gold standard for assessing a patient's infectivity, but it is a time-consuming technique and lacks sensitivity. SARS-CoV-2 subgenomic RNA (sgRNA) detection has been used as a proxy for assessing the infectivity but only a limited number of studies have described its use in vitro and in clinical samples. This study aimed to evaluate the correlation between results from viral culture, genomic RT-PCR (gRT-PCR), and subgenomic RT-PCR (sgRT-PCR) during in vitro infection and in clinical samples. In vitro viral replication kinetics showed that both genomic RNA (gRNA) and subgenomic RNA (sgRNA) levels remained stable up to 21 days in the absence of replication-competent virus. Using clinical samples, sgRNA was detected in 87.5% of culture-positive samples, demonstrating better performances than gRT-PCR (Positive predictive value (PPV) 93.3% and Negative predictive value (NPV) of 87.5%) and an almost perfect agreement with culture results (Cohen κ = 0.81 [95% CI: 0.66-0.95]). These findings suggest that testing for sgRNA and/or using a gRNA Ct cut-off of 21.2 could be used as a proxy to determine the presence of SARS-CoV-2 replication-competent virus.