The chiral resolving ability of the amylose-based Chiralpak IA chiral stationary phase towards omeprazole and other proton pump inhibitors under reversed-phase conditions was investigated. Organic modifier-buffer demonstrated to be a valid alternative elution mode with respect to conventional polar organic and normal-phases. No evidence of deterioration of performance of the enantioselective column after several multimodal cycles of elution was observed. Mobile phase composition was systematically changed in order to modulate the enantiomer elution order of set of compounds studied. A very simple method based on on-line detection of optical rotational sign during enantioselective HPLC was developed to assign the absolute configuration and enantiomeric elution order.