A simple gradient reversed-phase high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of total benzalkonium chloride (BAC) and pramoxine HCl in wound care solutions. Laboratory formulations were diluted 1 : 10 with water and injected into a HPLC system equipped with a Phenomenex, Luna CN column (100 Å, 5µm, 250 mm × 4.6 mm) in order for BAC and pramoxine to separate from other excipients and be detected by a UV detector (λ = 262 nm). The mobile phase was 0.075 mM sodium acetate trihydrate buffer (pH = 5.0) using multi-ramp gradient elution with acetonitrile. Quantitation was achieved by direct comparison of the peaks of BAC and pramoxine HCl of the sample to a reference standard of known concentrations. A stress study with acid, base, peroxide, heat, and light indicated no interference from drug product or excipients. The mean recovery results for both pramoxine and benzalkonium chloride at 100% level were 100.5 ± 0.3%, 100.5 ± 0.1% respectively (mean ± SD, n = 6). In this report, the full experimental results from developing and validating this method are presented.