A rapid and reliable analytical method has been developed to quantify the melatonergic antidepressant agomelatine in three matrices, and namely saliva, plasma and dried blood spots. The method is based on the use of liquid chromatography with fluorimetric detection exploiting the native fluorescence of agomelatine. For saliva and plasma samples an original microextraction by packed sorbent procedure was implemented obtaining satisfactory extraction yield of the analyte (always higher than 89%) and a good clean-up of the matrices. On the contrary, agomelatine was extracted from dried blood spots by suitable solvent microwave-assisted extraction and injected into chromatographic system. Satisfactory results in terms of sensitivity, linearity, precision, selectivity and accuracy were obtained. Thus, the developed method seems to be suitable for therapeutic drug monitoring of depressed patients under agomelatine therapy.