2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki= 250 μM (95% CI: 106-405 μM) and dose-dependently increased the rate of the ATP hydrolysis with KM= 112 μM (95% CI: 28-195 μM). The kinetic scheme consistent with this experimental data is proposed.