Optical photothermal infrared (OPTIR) spectroscopy and imaging were combined with fluorescence microscopy to track infection progression. Baculovirus infection in Spodoptera frugiperda (Sf) 9 cells with mCherry as a fluorescence imaging reporter was analyzed 3, 6, 12, and 24 h postinfection (hpi). Cells were sandwiched between two CaF2 windows separated by a thin spacer, forming a sealed aqueous chamber that maintained hydration for 4−6 h, enabling IR spectral acquisition and imaging of living cells throughout. Fluorescence microscopy demonstrated a distinct onset of the mCherry reporter signal beginning 12 hpi. Early infection time points 3 and 6 hpi showed increased protein (amide II, 1545 cm−1) and asymmetric phosphate (1241 cm−1) bands, while 12 hpi, the symmetric phosphate (1087 cm−1) band profoundly reversed, consistent with nucleic acid changes associated with infection progression. The amide II to nucleic acid ratio (S1545/1086), obtained from discrete wavenumber imaging, tracked aggregate infection-associated protein changes (of which mCherry is a late-stage contributor), initially increasing 6 hpi, decreasing 12 hpi, and reaching its maximum value 24 hpi. The fluorescence reporter was absent at early stage infection (3 and 6 hpi), where IR spectroscopy and imaging successfully discriminated infected from uninfected cell populations. Fluorescence imaging validated IR measurements for later infection time points (12 and 24 hpi). Infected cells were classified from negative controls with balanced accuracies up to 90%. Overall, this approach identifies spectral signatures consistent with viral infection, providing a labelfree method to discriminate infected populations from controls within their natural cellular environment.