The combined use of 3D-fluorescence spectroscopy and independent component analysis using a differential fingerprinting approach has been applied with success to detect physiological effects of dimethoate in honeybees. Biochemical determinations combined with the identification of fluorescence zones that may correspond to proteins, NADH or neurotransmitters/neurohormones (octopamine, dopamine and serotonin) related to the physiological stress caused by the pesticide enabled phenomenological modeling of the physiological response in the honeybee using a simple and rapid method. The signals associated with the fluorophores involved in the response to stress were extracted from the fluorescence spectra using an unsupervised algorithm such as independent component analysis. The signals of different neurotransmitters were isolated on separated factorial components, thus facilitating their biochemical interpretation.