This study investigates the proton sponge hypothesis, which suggests that cationic polymer-DNA complexes (polyplexes) enhance transgene delivery by increasing endosomal chloride (Cl⁻) accumulation and osmotic swelling/lysis. The hypothesis was tested by comparing the effects of three polyamines—polysine (POL), polyethylenimine (PEI), and polyamidoamine (PAM)—on endosomal Cl⁻ concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and these polyamines. Fluorescently labeled polyamines were used to measure Cl⁻ and pH in endosomes. Results showed that endosomes containing PEI or PAM polyplexes had significantly higher Cl⁻ accumulation and slower acidification compared to those containing POL polyplexes. Endosomes with PEI or PAM also swelled more and lysed later than those with POL. These effects were attributed to the greater H⁺ buffering capacity of endosomes containing PEI or PAM, which allowed for increased Cl⁻ entry and osmotic swelling. The study also demonstrated that endosomes containing PEI or PAM polyplexes were more sensitive to osmotic lysis than those containing POL polyplexes. This was confirmed by in vitro osmotic challenge experiments, where endosomes containing PAM polyplexes showed greater permeabilization and lysis compared to those containing POL polyplexes. The findings provide direct support for the proton sponge hypothesis, suggesting that the enhanced Cl⁻ accumulation and osmotic swelling in endosomes containing PEI or PAM polyplexes facilitate the escape of polyplexes from endosomes, leading to more efficient gene delivery. The study also highlights the importance of endosomal pH and Cl⁻ regulation in the efficiency of non-viral gene transfer. The results suggest that designing non-viral vectors with enhanced endosomal buffering capacity could improve the efficiency of gene delivery.This study investigates the proton sponge hypothesis, which suggests that cationic polymer-DNA complexes (polyplexes) enhance transgene delivery by increasing endosomal chloride (Cl⁻) accumulation and osmotic swelling/lysis. The hypothesis was tested by comparing the effects of three polyamines—polysine (POL), polyethylenimine (PEI), and polyamidoamine (PAM)—on endosomal Cl⁻ concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and these polyamines. Fluorescently labeled polyamines were used to measure Cl⁻ and pH in endosomes. Results showed that endosomes containing PEI or PAM polyplexes had significantly higher Cl⁻ accumulation and slower acidification compared to those containing POL polyplexes. Endosomes with PEI or PAM also swelled more and lysed later than those with POL. These effects were attributed to the greater H⁺ buffering capacity of endosomes containing PEI or PAM, which allowed for increased Cl⁻ entry and osmotic swelling. The study also demonstrated that endosomes containing PEI or PAM polyplexes were more sensitive to osmotic lysis than those containing POL polyplexes. This was confirmed by in vitro osmotic challenge experiments, where endosomes containing PAM polyplexes showed greater permeabilization and lysis compared to those containing POL polyplexes. The findings provide direct support for the proton sponge hypothesis, suggesting that the enhanced Cl⁻ accumulation and osmotic swelling in endosomes containing PEI or PAM polyplexes facilitate the escape of polyplexes from endosomes, leading to more efficient gene delivery. The study also highlights the importance of endosomal pH and Cl⁻ regulation in the efficiency of non-viral gene transfer. The results suggest that designing non-viral vectors with enhanced endosomal buffering capacity could improve the efficiency of gene delivery.