Gene therapy has emerged as a promising technique to treat many chronic diseases, genetic disorders and even cancer. Furthermore, the recent discovery that RNA interference could be used as an approach to modulate protein expression in mammalian systems sparked a potential revolution in disease treatment. By taking advantage of this endogenous mechanism, gene silencing can be induced by sequence-specific cleavage of a messenger RNA coding for a specific protein, by means of a short interfering RNA (siRNA). Introducing siRNA into cells is limited by numerous challenges, predominantly the lack of effective delivery systems that can safely transport these macromolecules to their site of action while overcoming multiple barriers that hinder the delivery pathway. For siRNA delivery, synthetic vectors, including polymers, have increasingly gained attention primarily due to their easily controllable molecular composition. In this work, we employed a combinatorial chemistry approach for the rational design of polymers as non-viral delivery systems. We hypothesize that by developing libraries that correlate the structure-function relationship of polymeric vectors, optimal structural parameters will be identified for efficient transport and delivery of siRNA into cells. Toward this goal, we carried out the controlled synthesis of poly(methacrylic acid) (pMAA) and poly(acrylic acid) (pAA) by reversible addition-fragmentation chain transfer (RAFT) polymerization. Based on these two polymer precursors, we evaluated the conjugation of two ligands, D-(+)-galactosamine (Gal) and agmatine (Agm) to generate polymer libraries by varying the binary subtitution of both moieties. Due to the higher Agm/Gal side chain substitution in pAA as compared to pMAA, pAA conjugates were selected for in vitro evaluation. pAA was synthesized with four distinct molecular weights (Mn), specifically 3 kDa (PDI = 1.36), 5 kDa (PDI = 1.32), 10 kDa (PDI = 1.19) and 21 kDa (PDI = 1.19). For each polymer Mn, various combinations of Agm and Gal were substituted, for a total of 22 polymers under evaluation. From the biophysical and cellular characterization, it was determined that both the Agm/Gal content and the Mn significantly influence the ability of these polymer conjugates to serve as siRNA delivery systems. As distinguished from these analyses, the higher the Agm content, the more compact and stable the polyplexes and the higher transfection efficiency, but also the higher cytotoxicity. As for the effect of molecular weight, the lower the Mn, the more stable the polyplexes and the lower cytotoxicity, but also the lower transfection efficiency. Therefore, a critical balance between Agm/Gal content and polymer Mn must be attained to acheive favorable outcomes - these being high transfection efficiency with low cytotoxicity. The best candidate identified was 5-P3, corresponding to a pAA Mn = 5 kDa, and Agm and Gal contents of 55% and 17%, respectively. The development of polymer libraries and their in vitro evaluation offered a better understanding of the structure-function relationship of polymeric vectors. This approach provided the identification of optimal structural parameters and served as a synthetic foundation upon which safer and more efficient siRNA delivery systems can be developed.