Using TEM, we first examine the morphologies of LPEI and BPEI polyplexes, followed by UV-Vis absorbance spectroscopy to analyze the complexation kinetics of these polymers to DNA. We then apply Fourier Transform IR (FTIR) spectroscopy with Multivariate Curve Resolution (MCR) analysis to differentiate between the binding modes of LPEI and BPEI polyplexes. Our comprehensive approach elucidates new insights into the molecular-level binding mechanisms of PEI systems to DNA, which we anticipate will help guide the development of more efficient and versatile PEIbased gene delivery systems.