This paper describes the ability of the combination of electrospray ionization mass spectrometry (ESI-MS) and anion-exchange chromatography coupled with inductively coupled plasma atomic emission spectrometry (AEC-ICP-AES) for cobalt speciation study in the binary cobalt-cysteine system. ESI-MS, allowing the identification and the characterization of the analytes, is used as a technique complementary to AEC-ICP-AES, providing elemental information on the separated species. The methods have been developed through the study of samples containing Co2+ and 1-fold to 5-fold molar ratios of cysteine over a pH range 2.5 to 11. In each case, cobalt-cysteine complexes were characterized by ESI-MS in negative ion mode. AEC-ICP-AES allowed further separation and detection of the cobalt species previously characterized. The strong influence of pH and ligand-to-metal ratios on the nature and stoichiometry of the species is demonstrated. For the first time, a direct experimental speciation diagram of cobalt species has been established owing to these analytical techniques. This work is a promising basis for the speciation analysis of cobalt, since a good knowledge of cobalt speciation is of prime importance to better understanding its fate in biological and environmental media.