This paper describes the development of an analytical method for the determination of two pseudo-oligosaccharides, acarbose and its main metabolite. The analysis was carried out by liquid chromatography on a porous graphitic carbon stationary phase. The separation mechanism of these compounds is dominated by charge-induced interactions between the polar analyte and the polarizable surface of graphitic carbon. Several chromatographic parameters were investigated, including the nature and percentage of the organic solvent and acid modifier as well as column temperature. The best conditions were achieved with a mobile phase containing trifluoroacetic acid 0.1% and acetonitrile 8%. Under these conditions, the simultaneous resolution of acarbose and its metabolite as well as of their anomers was achieved, highlighting the potential of this stationary phase for challenging separations. Moreover, three detection methods are described and compared in terms of sensitivity. The evaporative light scattering detector was not sensitive enough for acarbose and metabolite determination in biological fluids. Mass spectrometry allowed a significant improvement in sensitivity but was not sufficient to permit the analysis of selected compounds in such complex matrices. However, this detector confirmed the presence of acarbose and its metabolite as well as the anomeric separation. Finally, fluorescence detection was found to be very sensitive after derivatization with 2-aminobenzamide and enabled the determination of selected compounds at a low concentration level.