The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivatedthe search and improvement of optimized purification processes. In this context, dipeptides l-tyrosine-l-tyrosine and l-tyrosine-l-arginine are synthetized to explore their application as affinity ligands forsupercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N-Boc-l-tyrosine, followed by condensation with l-tyrosine or l-arginine methyl esters in the presence ofdicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides.The supports are then obtained by coupling l-tyrosine, l-tyrosine-l-tyrosine and l-tyrosine-l-arginineto epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS)NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor isused to establish the promising ligand to be used in the chromatographic experiments and ascertainexperimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln)pDNA, being the open circular (oc) the one that promoted the lowest affinity to l-tyrosine-l-arginine.Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobicwith the majority of the 5'-mononucleotides, except for 5'-GMP with l-tyrosine-l-arginine Sepharose that is mainly electrostatic. The support l-tyrosine Sepharose used in chromatographic experiments pro-motes the separation of native pVAX1-LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing thesalt concentration. The results suggest that it is possible to purify different plasmids with the l-tyrosineSepharose, with slight adjustments in the gradient conditions.