During the COVID-19 pandemic, the standard diagnostic assay for SARS-CoV-2 detection was RT-qPCR using TaqMan probes, with samples primarily taken through nasal and oropharyngeal swabs. The TaqMan-based method is costly, highlighting the need for a more affordable alternative for SARS-CoV-2 diagnosis. As an alternative strategy, we developed and evaluated a SYBR Green-based RT-qPCR method targeting the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. Under optimized RT-qPCR conditions, the sensitivity and linearity of the SYBR assays were assessed by using in vitro-transcribed RNA and RNA extracted from cultured SARS-CoV-2 isolates of the Wuhan reference strain and various circulating variants. Our results demonstrated that the SYBR Green-based RT-qPCR method was successfully developed with sufficient performance. The assay could detect up to 25 copies of in vitro-transcript RNA per reaction. Meanwhile, using the RNA extracted from cultured virus, the SYBR green assay was able to detect virus concentrations at least as low as 1 PFU/mL per reaction for all the variants tested. When tested on clinically relevant samples (88 naso-oropharyngeal swabs and 47 saliva samples), comparable results with the TaqMan assay were demonstrated. The Ct values of both methods for the positively detected samples were similar, with a difference in Ct of 0.72 ± 0.83 (p = 0.392) and −0.7765 ± 0.6107 (p = 0.209) for naso-oropharyngeal swab and saliva samples, respectively. These findings suggest that the SYBR method is reliable and thus offers an alternative assay for the detection of SARS-CoV-2. In particular, using saliva specimens could allow this assay to serve as a simple approach for SARS-CoV-2 detection. © 2025 by the authors.