A mixed-polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric beta-CD, 0.4% w/v tetronic (R) 1107 and 400 mM trisphosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 50 cm of total length and 75 mu m id, an applied voltage of -7 kV, a temperature of 30 degrees C and 200 nm UV-detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 mu g/mL) (OSCS) and 0.1% w/w (0.1 mu g/mL) (Der), and values of LOQ 0.2% w/w (0.2 mu g/mL) (OSCS) and 0.3% w/w (0.3 mu g/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.