Global resurgence of human and animal poxviral diseases calls for diagnostic innovation. Here we explored a novel strategy to quantitate poxvirus infection in living cells through an integrated reporter system specifically activated by infectious particles. Combining constitutive expression of green fluorescence protein (gfp) transcripts flanked by poxvirus transcriptional elements and retrovirus reverse transcriptase (RT), we developed a continuous stable cell line where fluorescence was driven by poxvirus transcriptional machinery upon recognition of reverse-transcribed gfp transcripts in the cytosol. Constitutively expressed gfp transcripts were reverse-complemented to prevent direct translation in the absence of poxvirus infection. Maloney murine leukaemia virus (MMLV) RT was selected for its exclusively cytosolic localisation and superior activity. GFP-positive cells were detected only in the presence of reporter cassette and RT and upon infection with vaccinia, cowpox and ectromelia viruses, but not herpes simplex virus, confirming specificity for poxviruses. RNA FISH experiments revealed that only cells with high positive-sense gfp transcript copy number turned GFP-positive, suggesting a threshold determined by RT activity during poxvirus infection. Nonetheless, positivity was dependent on genome replication, suggesting detection of infectious virus. Our study integrates multiple biotechnological advances to timely pioneer a novel fluorescence-based strategy to diagnose and quantitate virus infection and provides proof-of-concept to produce a reporter gene in its cDNA form in the cytosol of cells, where poxviruses and asfarviruses replication takes place. Subsequent activation by live poxviruses offers distinctive advantages over other detection methods including the measurement of infectious virus in an automated system with minimal operating requirements.