We report for the first time the absolute quantification of a metalloprotein separated by nondenaturing gel electrophoresis (GE) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in combination with species-specific isotope dilution mass spectrometry (IDMS). The proposed method is based on the use of an isotopically enriched (57)Fe-transferrin complex to quantify natural transferrin (Tf) in human serum samples. First, the saturation process of Tf with natural abundance or isotopically enriched (57)Fe was accomplished by using freshly synthesized Fe-citrate solutions. The stability of the metal-protein complex as well as its stoichiometry was investigated by spectrophotometry and ICP-MS, demonstrating a satisfactory stability over a period of at least one month and a molar ratio Fe:Tf of 1.94 ± 0.09, which is close to the expected value of 2. The species-specific IDMS method was compared with external calibration using the Fe-Tf (absolute Tf amount between 2 and 10 μg) and different sample preparation procedures (stained and nonstained gels) as well as two laser ablation strategies (single line ablation in the direction perpendicular or horizontal to the electrophoretic migration) were evaluated. The proposed species-specific GE-LA-ICP-IDMS method was tested for the analysis of a serum certified reference material (ERM-DA470k/IFCC). The results were in good agreement with the certified value with relative standard deviation values in the range of 0.9-2.7% depending on the data treatment procedure used. Furthermore, the analysis time has been drastically reduced in comparison with previous approaches to less than 15 min. The quantification by species-specific GE-LA-ICP-IDMS allowed us to obtain accurate and precise results not only by analyzing the protein spot in the middle position but also in the adjacent ablation line to the center.