The fluorescence-based sensor used in the present study represents a rapid and non-invasive tool, already used for assessing grape phenolic maturity and quality, and recently tested also on plums (for maturity assessing) and turf (for nitrogen concentration). It may represent in perspective an alternative to destructive laboratory analyses, which are time-consuming and require an accurate sampling approach. Based on our knowledge, this is the first experience of its use on tobacco to test flavonols and nicotine derivatives in fresh and cured leaves. The activity carried out in 2012 on tobacco of Fattoria Autonoma Tabacchi of Città di Castello compared HPLC/DAD/MS analyses of the hydroalcoholic extracts of tobacco leaves for flavonols and nicotine derivatives with those collected by a portable fluorimeter sensor UV-Vos (Multiplex^®, Force-A). Various parts of leaves, belonging to different crowns, and tobacco varieties were tested. This work aimed at establishing any correlation between the sets of data under study. Presently, the activity has been transferred to field grown tobacco at Fattoria Autonoma Tabacchi of Città di Castello, to find any correlation between actual measurements and leaf maturity parameters. Preliminary data are presented on the activity in progress. In a short perspective, the use of this sensor is expected to become a tool to drive agronomic treatments and best crop harvest time.