A plaque assay, the gold-standard method for measuring replication-competent lytic virions, requires staining and >48 h runtime. This study demonstrates a combination of lens-free holographic imaging and deep learning to automate and expedite the assay. The compact device captures label-free phase information at ~0.32 gigapixels per hour per well, covering 30 × 30 mm² and a 10-fold larger dynamic range than standard assays, quantifying infected area and plaque-forming units (PFUs). For vesicular stomatitis virus (VSV), the assay detected initial cell lysis at 5 h and >90% PFUs at <20 h with 100% specificity. Incubation time was reduced by ~48 h for herpes simplex virus type 1 and ~20 h for encephalomyocarditis virus. This stain-free assay is suitable for virology research, vaccine development, and clinical diagnosis.
