Abstract <p>New modifiers of electrophoretic systems—cationic polyelectrolytes with micellar properties, i.e. poly-11-acryloyloxyundecyl-<i>N</i>-methylpiperidinium bromide (<b>pAUMP-Br</b>), poly-11-acryloyloxyundecylpyridinium bromide, poly-11-acryloyloxyundecyl-1,4-diazabicyclo[2.2.2]octane-1-ium bromide, and a chiral copolymer based on acylated quinine and <i>N</i>-(11-acryloyloxyundecyl)-<i>N</i>-methylpiperidinium bromide (<b>pAUMP–AQin</b>)—have been synthesized. The electrophoretic properties of these polymers have been studied in the separation model mixtures of biologically active substances, steroid hormones, biogenic amines, and amino acids, in micellar electrokinetic chromatography and capillary electrochromatography modes. It was found that the new polyelectrolytes are polyfunctional modifiers of quartz capillary walls and the background electrolyte and ensure the implementation of various modes of electrophoretic separation of biologically active substances, indirect detection in the presence of a chromophore group in the polymer, generation of an anodic electroosmotic flow, and governing the efficiency and selectivity of the analyte separation. Field-amplified sample stacking in a quartz capillary modified with pAUMP-Br was performed, which ensured the 15-fold preconcentration of biogenic amines and reduced the limits of detection. It was found that the pAUMP–AQin copolymer with a chiral label favors the separation of tryptophan enantiomers.</p>