A novel and non-invasive sampling method was developed for use in electrospray ionization/mass spectrometry (ESI/MS). The sampling tool, a watercolor pen (brush) that had been rinsed with ethanol, was used to collect analytes from the eyelids of volunteers. Following this, the brush was quickly moved to the front of the mass inlet, where the ethanol as well as the analytes evaporated very fast and escaped from the brush surface. When the analytes make contact with the ESI plume, which arises from the ESI needle tip, they are ionized and then detected by a mass spectrometer. The findings show that this new technique is applicable for the analysis of caffeine and its metabolites in samples obtained from eyelids after the volunteers consumed coffee. The methodology is simple and economical, non-invasive, and is suitable for use in monitoring the levels of caffeine and its metabolites over an extended period of time.