The chromatographic properties of new N,O-dialkyl phosphoramidate stationary phases were studied. Obtained stationary phases containing C10 (Amino-P-C10) and C18 alkyl chains (Amino-P-C18) were attached to aminopropyl silica using the phosphate group. As basic characteristics of the bonded phases, the hydrophobicity and silanol activity (polarity) were investigated. Columns were compared according to the methylene selectivities using a series of alkylbenzene homologues and according to their shape and size selectivities using polycyclic aromatic hydrocarbons (PAHs). The measurements were done using methanol (MeOH)-water and acetonitrile (ACN)-water mobile phases. The presented results show that the coverage density of the bonded ligands and the length of the alkyl chains strongly influence the retention and selectivity of the N,O-dialkyl phosphoramidate bonded phases in reversed-phase liquid chromatography. The presence of the polar amino and phosphoramidate groups in the structure of the bonded ligands strongly influence the polarity of the bonded phase. A higher hydrophobicity is exhibited by the Amino-P-C18 adsorbent. The chromatographic properties of the new stationary phases were compared with octadecyl, cholesterol-bonded and immobilized artificial membrane (IAM) stationary phases.