The degradation of DNA in lysosomes represents a major obstacle to efficient nonviral gene delivery. The rational design of vectors that overcome this obstacle requires a better understanding of the lysosomal barrier to gene delivery, which in turn requires a means to investigate this intermediate step. To this end, we developed a technique to measure the pH environment of delivered DNA, from which the degree to which vectors avoided trafficking to acidic lysosomes could be determined. The measured average pH of DNA delivered using poly-L-lysine (PLL) polyplexes was 4.5, suggesting that PLL polyplexes were trafficked to acidic lysosomes. Other vectors could avoid or buffer the pH of lysosomes as DNA delivered using Lipofectamine Plus, polyethylenimine (PEI), linear polyethylenimine (LPEI), and two degradable poly(beta-amino ester)s (poly-1 and poly-2) had average pH values of 7.1, 5.9, 5.0, 6.7, and 6.4, respectively.