An HPLC procedure on a polar reversed-phase column with mobile phases containing ionic liquid (IL) was developed for the analysis of selected alkaloids from different chemical groups. We aimed to obtain optimal conditions for the separation of alkaloids because widely used silica-based stationary phases exhibit a silanol effect, rendering analysis of basic analytes extremely difficult. Retention, separation selectivity, peak symmetry, and system efficiency were examined in various eluent systems containing different concentrations of IL and acetonitrile. The obtained results revealed substantial influence from the concentrations of IL, the organic modifier, and temperature on the retention behavior of the investigated alkaloids. The most selective and efficient chromatographic systems were applied for the analysis of several alkaloids in a plant extract.