A simple and rapid RP-LC method was developed for routine analysis of benzalkonium chloride (BKC) and its impurities in benzalkonium chloride raw material. The method uses a short ACE 3 C8 column (50 × 4.6 mm, 3 μm particle size) and a gradient mobile phase consisting of 0.025% trifluoroacetic acid in water/acetonitrile (90/10, v/v) as mobile phase A, and water/acetonitrile (30/70, v/v) as mobile phase B. The four commonly observed homologues (i.e., C10, C12, C14 and C16) that are present in typical commercial lots of BKC are well separated from each other in less than 21 min. This method has been demonstrated to be able to simultaneously separate and quantitate at parts-per-million levels the three potential impurities (i.e., benzyl alcohol, benzaldehyde and benzyl chloride) in commercial lots of BKC raw material. In addition, this method has the potential to be readily adopted or adapted for determining BKC homologues in a finished drug product; the feasibility of the latter capability is demonstrated through an artificial product solution containing a model API and typical excipients used for liquid formulation drug products.