Murine gammaherpesvirus 68 (MHV-68) provides a valuable tool to screen novel therapeutic strategies against oncogenic gammaherpesviruses. The development and characterization of antiviral agents usually depend on appropriate screening assays. The aim of this study was to develop rapid and sensitive method for testing antiviral compounds against gammaherpesviruses. For this purpose, a recombinant MHV-68 expressing firefly luciferase (MHV-68/LUC) was constructed. The conditions for MHV-68/LUC infection in Vero cells suitable for novel antiviral screening assay in 96-well plate format were then optimized. The sensitivity of MHV-68/LUC to acyclovir (ACV) and ganciclovir (GCV) was measured by the optimized luciferase activity reduction assay. The 50% inhibition concentration (IC50) values for ACV and GCV were comparable to those determined by conventional plaque reduction assay. Therefore, the luciferase activity reduction assay can efficiently replace the plaque reduction assay. The great advantages of novel assay are represented by the significant reduction in assay time and rapid and objective measurement of the assay. In order to evaluate whether the luciferase activity reduction assay could be used as a screening system for novel antivirals, newly synthesized quinolone/quinoline derivatives were tested for their effects on the replication of MHV-68/LUC in vitro. The compound 2-(1-(b-D-Xylopyranosyl)-1,2,3-triazol-4-yl)-3,4-dibenzyloxy-quinoline showed significant antiviral activity and its IC50 against MHV-68/LUC was estimated to be 1,76 µg/ml. However, this compound was not suitable for in vivo testing due to its narrow selectivity index (SI = 11). Keywords: MHV-68; antiviral screening; luciferase; quinolone/quinoline derivatives.