To evaluate the effects of antipruritic drugs, it is important to determine whether the neural responses induced by physiological itch stimuli are suppressed. Although there are several behavioral assessments for topical antipruritic drugs applied to the skin, there are few established methods at neuronal levels using in vivo electrophysiological recordings for predicting local efficacy of antipruritic drugs for cutaneous application. To establish an assessment of topical antipruritic drugs applied to skin using in vivo extracellular recording from neurons in the superficial dorsal horn, we examined the relationships between itch-related biting behavior and spinal neuronal responses elicited by intradermal injection of pruritogen serotonin (5-HT) in hairless mice. The efficacy of topical occlusive application of local anesthetics was also evaluated by an in vivo electrophysiological method. 5-HT significantly increased the firing frequency in spinal neurons. The spinal firing frequency time course was similar to that of the biting behavior after the 5-HT injections. The 5-HT-induced spinal responses were significantly decreased by topical occlusive application of lidocaine or a Nav 1.7 channel blocker to the calf. The intradermal 5-HT injection-induced spinal neuronal responses appeared to be suppressed by topical occlusive application of lidocaine or a Nav1.7 channel blocker. The electrophysiological method for evaluating topical antipruritic drugs may be beneficial in assessing local effects on the skin.