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The Caribbean spiny lobster Panulirus argus supports important fisheries throughout the greater Caribbean and is also the only known host for the pathogenic virus Panulirus argus virus 1 (PaV1). While discovered nearly 2 decades ago, gaps still exist in our knowledge of PaV1, such as the dose required to establish infection and its viability outside of the host. To help answer such questions and to enhance diagnostic capabilities, we developed a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay for PaV1. Of the advantages offered by qPCR, one of the most important benefits is its ability to accurately quantify viral DNA copies in a clinical sample. The qPCR assay was found to be efficient (mean ± SD: 99.19 ± 4.67%) and sensitive, detecting as few as 10 copies of PaV1 plasmid DNA. Its diagnostic sensitivity and specificity determined using a set of 165 lobster samples (138 from Florida, USA, and 27 from across the Caribbean) were 100 and 84%, respectively. The qPCR assay should thus prove useful as a research tool and for detecting and quantifying PaV1 infection severity in Caribbean spiny lobsters.