Conditions for the separation and determination of benzalkonium chloride (BAC) homologues by CE with UV-detection and CE coupled to MS (IT) using electrospray as ionization source were established. The separation was performed using fused-silica capillaries of 50 μm id and 100 mM acetic acid-ammonium acetate buffer solution at pH 4.5 with 80% of ACN as carrier electrolyte. CE-MS coupling parameters were optimized and methanol-10 mM acetic acid (90:10 v/v) was selected as sheath liquid. Detection limits, based on an S/N of 3:1, were calculated, and values between 0.8 and 1.3 mg/L with CE-ESI/MS and around 0.5 mg/L with CE-ESI-MS/MS, using hydrodynamic injection (15 s, 3.5 kPa), were obtained. Good run-to-run and day-to-day precisions on concentration were achieved with RSDs lower than 8%. Quantitative analysis was carried out by the internal standard method and the calibration curves showed good linearities (r2>0.98). The CE-ESI-MS/MS method was successfully applied to the analysis of BAC in different ophthalmic solutions, allowing the direct determination, identification and confirmation of the BAC homologues presented in these samples.