A rapid, reproducible, and sensitive ion-exchange chromatography (IEC) method combined with graphite-furnace atomic absorption spectrometry (GF-AAS) was developed to separate and quantify basal amounts of both metallothionein (MT) isoforms in dab (Limanda limanda) liver samples. Dab liver homogenates were saturated with Cd, and obtained cytosols were purified by a two-step acetone precipitation prior to chromatographic analysis. Metallothionein isoforms were separated by IEC and were subsequently quantified indirectly by GF-AAS via their Cd contents. The amount of Cd needed for saturation was optimized. The efficiency of Cd saturation and acetone precipitation was proven by gel permeation chromatography (GPC) and metal distribution analysis. Based on the method of standard addition, a recovery for MT was 98% after acetone precipitation and 68% after IEC. The repeated determination of MT isoforms in a dab liver homogenate resulted in coefficients of variation of approximately 12% for both isoforms. Based on the detection limit for GF-AAS, the calculated detection limit for MT isoforms is 2 ng/mg protein. Therefore, this method is suitable for monitoring purposes. The implications of isoform-specific measurement for biomarker monitoring are discussed.