Objective To develop a rapid, high-sensitivity, and high-specificity assay based on TaqMan real- time fluorescent quantitative PCR (TaqMan real-time FQ-PCR) targeting the gag gene of simian-human immunodeficiency virus (SHIV) for quantitative measurement of simian immunodeficiency virus (SIV) and SHIV in plasma, peripheral blood mononuclear cells, and lymph nodes from rhesus macaques. Methods A 109-bp fragment of the SHIV_SF162p3n gag gene was amplified using reverse-transcription PCR and cloned into the pGEM? -T vector to construct the pGEM? -T-SHIV gag plasmid, which was used as a standard for the assay. Results TaqMan real-time FQ-PCR efficiently detected 10² – 10⁷ copies/ μL SIV/ SHIV gag mRNA with a correlation coefficient (R² ) of 0. 998 and slope of - 3. 304. The coefficient of variability was 0. 6%– 1. 1%. Conclusions This rapid assay exhibited both high sensitivity and high specificity. Thus, this method is suitable for studies involving SIV/ SHIV infection macaque models.