Metallothioneins (MTs) are ubiquitous and inducible proteins characterized by low molecular mass (Mr 6-8 kDa), high Cys content (20-30%) but no aromatic or His residues, and strong affinity to binding toxic metals (Cd, Hg, Ag, Pb) in metal-thiolate clusters. Due to their induction by a variety of stimuli, MTs are considered suitable biomarkers in the medical and environmental fields. The protective role of MTs from Cd toxicity and lethality is well-established. Although MT assessment is a difficult task, the accurate measurement of MT is mandatory in order to assess its biomarker potential and to identify new outstanding biological roles. We have developed a highly specific, sensitive, and reliable method for total MT quantification in unheated extracts by reversed-phase high-performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD). A derivatization protocol with monobromobinane, a thiol-specific fluorogenic reagent, is required after heat-, SDS-EDTA-and DTT-treatment. SDS-polyacrylamide gel electrophoresis was used to confirm the identity of the mBBr-labeled MT peak resolved by RP-HPLC-FD. The method has been successfully used to quantify MT content in the digestive gland of various clam species from Southern Spanish sites with different metal levels, and also in the liver of fish injected with different Cd, Cu and Hg doses. MT levels obtained by RP-HPLC-FD in non-heated extracts were significantly higher when compared to those obtained by other well-established assays relying on solvent precipitation (spectrophotometry) or heating (differential pulse polarography) pre-purification steps.