Kaposi’s sarcoma-associated herpesvirus (KSHV) has both latent and lytic phases of replication. The molecular switch that triggers a reactivation is still unclear. Cells from the S phase of the cell cycle provide apt conditions for an active reactivation. In order to specifically delineate the Raman spectra of cells supporting KSHV reactivation, we followed a novel approach where cells were sorted based on the state of infection (latent versus lytic) by a flow cytometer and then analysed by the Raman tweezers. The Raman bands at 785, 813, 830, 1095 and 1128 cm−1 are specifically altered in cells supporting KSHV reactivation. These five peaks make up the Raman fingerprint of cells supporting KSHV reactivation. The physiological relevance of the changes in these peaks with respect to KSHV reactivation is discussed in the following report.