Cationic lipid-mediated gene transfer represents a promising approach for the treatment of a number of diseases. Since the successful introduction of DOTMA:DOPE (Lipofectin), a variety of cationic lipids have been developed for use in gene transfer. Some of the more active cationic lipid formulations, including GL-67:DOPE, DC-chol:DOPE, DMRIE:DOPE and DOTAP, have been used in human clinical trials. It is of critical importance to develop robust analytical methods for the determination of the chemical purity of these formulations. We report here efficient, sensitive, and reproducible reversed-phase HPLC methods for use in determining the chemical purity of cationic lipid formulations. GL-53:DOPE, GL-67:DOPE, DMRIE:DOPE, DC-chol:DOPE, GAP-DLRIE:DOPE, DOTMA:DOPE (Lipofectin), DDAB:DOPE (Lipofectace), DOSPA:DOPE (Lipofectamine), DOGS (Transfectam), and DOTAP were analyzed by HPLC on C8 or C18 bonded phase columns with aqueous/mixed organic mobile phases containing trifluoroacetic acid and with ELSD detection in the gradient elution mode. Baseline resolution of the components of the GL-53:DOPE formulation was achieved by optimization of the solvent system and gradient profile. Capacity factors (k') of the cationic lipids were greatly affected by the end-capping chemistry of the C18 bonded phases. The calibration curves for GL-53, DC-chol, DMRIE, and DOPE were determined in the range of 1.6–200.0 μg. The detection limits for these compounds were determined to be 0.4–1.6 μg