An major research area in environmental chemistry is the development of methods for the analysis of biomarkers. Metallothioneins are used as biomarkers in studies of heavy metals exposure in water, because metallothioneins are synthesized and accumulated when organisms are exposed to toxic concentrations of pollutants. In this work, simple and sensitive voltammetric methods were developed for metallothionein and copper (II) determinations in fish liver Lepomis gibbosus. Both analytical methodologies were optimized and applied to samples extracted from individuals previously submitted to sub-lethal toxicological trials with copper sulphate (CuSO 4 ) and cadmium chloride (CdCl 2 ). The obtained results showed that both methods are very precise, sensitive, and involve simple sample preparation processes. Moreover, metallothioneins showed better correlation with the toxic exposure than Cu 2? . To the best of our knowledge, this is the first time that hepatic metallothioneins and Cu 2? contents are voltammetrically determined in order to be compared in their function as heavy metal biological indicators.