Amphiphilic block copolymers containing poly(2-vinyl pyridine) (P2VP) as a hydrophilic part and polystyrene (PS) as a hydrophobic segment have a number of applications in modern technology. The presence of a lone pair of electrons on the pyridine moiety of P2VP is exploited for many important applications which on the other hand creates problems in molecular characterization of these polymers by chromatographic methods. P2VP is particularly considered as a unique case for liquid chromatography because of the presence of different types of unwanted polar interactions with most of the stationary phases. Hence, the scientific reports on liquid chromatographic analysis of P2VP homo- and copolymers are scarce. The current study is dedicated to develop a chromatographic characterization method for P2VP-containing copolymers. The major challenge in this context was to develop a system that allows systematic switching of elution behavior of P2VP as a function of mobile phase composition. Chromatographic critical conditions of P2VP are established for the first time on a NP column. The adequacy of the developed method is validated by analyses of blends and block copolymers of P2VP with PS and poly(methyl methacrylate). Further, chromatographic critical conditions of PS are established on a reversed phase column to evaluate block length of the P2VP block of PS-b-P2VP. The proposed method successfully demonstrates its capability to separate both types of homopolymers from block copolymers alongside estimation of block length of the noncritical segment.