The aim of this study was to assess the retention and selectivity of a cocktail of 10 substrates of uridine diphosphate glucuronosyltransferase enzymes (UGTs) and their respective glucuronides using four chro-matographic approaches. For this purpose, seven different stationary phases were employed in reversedphase liquid chromatography (RPLC), two in hydrophilic interaction liquid chromatography (HILIC), onein aqueous normal phase chromatography (ANPC) and four in subcritical fluid chromatography (SFC).Highly orthogonal separations were achieved with these chromatographic modes. Hydrophobic interac-tions mainly governed the retention of the substrates and their polar glucuronides in RPLC despite theuse of different chemical stationary phase bonding, involving additional possible interactions. In ANPC,atypical separations and poor peak shapes were observed with the selected compounds. In HILIC andSFC conditions, the metabolites were more retained than the substrates because of the polarity increaserelated to the glucuronic acid moiety. For the latter, a very high proportion of organic solvent (up to 80%)was required to elute the glucuronides that often displayed poor peak shapes. Finally, the selectivity ofnine chromatographic systems was compared for the separation of isomeric and diastereoisomeric com-pounds. The stationary phases used in RPLC mode were more selective towards the two positional isomersof morphine glucuronides since they possess distinct lipophilicity. HILIC and SFC columns were found tobe promising for the separation of a critical diastereoisomers pair, namely epitestosterone-glucuronideand testosterone-glucuronide.