Voltage-gated sodium channels (Nav) are molecular targets of clinically used drugs for treatments of various diseases (epilepsy, chronic pain, cardiac arrhythmia...) and also of numerous animal and plant neuro- toxins. The development of easy-to-use screening assays for the search of new ligands from chemicals libraries, animal venoms or plant extracts represents a challenge of a great interest to generate therapeutic hits. Here, we used the mammalian GH3B6 pituitary cell line, which consti- tutively expresses three different neuronal Nav channels isoforms (Nav1.2, Nav1.3 and Nav1.6), to identify novel compounds of pharmaco- logical interest from a library of in-house natural alkaloids. For this screening, we developed a method based on the use of Voltage-Sensor Probes (VSPs) that we adapted to detect both activators and blockers of Nav channels. Among the 62 alkaloids tested, 11 appeared as potent Nav channels inhibitors. Five other compounds were characterized as specific gating modifiers. While most of these alkaloids are already described in the literature, their ability to modulate Nav channels was unknown. In conclusion, we report here the suitability of this novel VSPs-based screening method i) to challenge the discovery and ii) to assess the ac- tivity of novel ligands on Nav channels.