To the best of our knowledge enantioselective chromatographic protocols on β-amino acids with polysaccharide-based chiral stationary phases (CSPs) have not yet appeared in the literature. Therefore, the primary objective of this work was the development of chromatographic methods based on the use of an amylose derivative CSP (Lux Amylose-2), enabling the direct normal-phase (NP) enantioresolution of four fully constrained β-amino acids. Also, the results obtained with the glycopeptide-type Chirobiotic T column employed in the usual polar-ionic (PI) mode of elution are compared with those achieved with the polysaccharide-based phase. The Lux Amylose-2 column, in combination with alkyl sulfonic acid containing NP eluent systems, prevailed over the Chirobiotic T one, when used under the PI mode of elution, and hence can be considered as the elective choice for the enantioseparation of this class of rigid β-amino acids. Moreover, the extraordinarily high α (up to 4.60) and R S (up to 10.60) values provided by the polysaccharidic polymer, especially when used with camphor sulfonic acid containing eluent systems, make it also suitable for preparative-scale enantioisolations.