Most HPLC enantiomer separations are performed with columns packed with a chiral stationary phase (CSP) operated with an achiral mobile phase. The intrinsically limited chemical selectivity of most CSPs to the simultaneous resolution of several pairs of enantiomers means that complex mixtures of diverse pairs of enantiomers cannot be resolved in a single run due to peak overlapping. Moreover, some drawbacks remain when the analyte is present in very complex samples containing other achiral compounds which can co-elute with the enantiomer peaks. Multidimensional chromatography becomes an option to increase peak capacity and resolve these samples. The aim of this work was to study an online fully comprehensive 2D-LC mode utilizing a combination of a chiral column in the first dimension and an achiral column in the second dimension. The 2D-LC system was built with an active flow splitter pump in order to easily adjust the volume of sample transferred into the second dimension and to independently optimize the flow rate in the first dimension. The present LCxLC method was optimized for the separation of amino acids present in honey samples, taking into account key parameters that influence the bidimensional peak capacity (orthogonality, sampling frequency, etc.). The amino acids have been preconcentrated on a cation-exchange column followed by derivatization. Several amino acids present in different honey samples have been identified and the data generated has been analyzed by principal component analysis.