Development of a feasible method for studying the competitive interaction between a pair of antagonists is essential for understanding the antagonism of trace metals in biological systems. Herein, we report the application of CE on-line coupled with ICP mass spectroscopy (CE-ICP-MS) to investigate the competitive binding of Zn2+ against Cd2+ for glutathione (GSH), which is related to the detoxification of Cd2+ in biological system, and introduce a method to evaluate the kinetics and thermodynamics for the competitive binding of Zn2+ against Cd2+ for GSH. The CE-ICP-MS hybrid technique allows easy and sensitive probing of the competitive binding of Zn2+ against Cd2+ for GSH and quantitative determination of the important thermodynamic and kinetic parameters of the competitive binding of Zn2+ against Cd2+ for GSH. Owing to the high sensitivity and element selectivity with multi-elements detection capacity of ICP-MS, we detailed the evaluation of the kinetics and thermodynamics describing the competition of Zn2+ against Cd2+ for GSH from the systematic data obtained by CE-ICP-MS. The competitive binding of Zn2+ against Cd2+ for GSH was demonstrated exothermic and thermodynamically favorable (Delta G = -7.2 kJ/mol) and driven entirely by a large favorable enthalpy decrease (Delta H = -15.1 kJ/mol) but with an unfavorable entropy decrease (Delta S = -25.6 J/mol/K). The kinetic data were fit to a second-order equation with the reaction rate constant (k) of (2.18 +/- 0.10) x 10(2) L/(mol . s) under the simulated physiological condition.