A method for benzalkonium analysis has been developed to measure benzalkonium concentration in dissolved and particulate fractions from urban runoff samples. The analysis was performed by liquid chromatography coupled with mass spectrometry (LC-MS/MS). The dissolved matrix was extracted by Solid Phase Extraction (SPE), with cationic exchange and the particles by microwave extraction with acidified methanol. Recovery percentages were closed to 100% for benzalkonium C12 and C14. The protocol was applied to roof runoff samples collected after a roof demossing treatment, and to separative stormwater samples from a 200 ha catchment. The results illustrate an important contamination of the roof runoff, with a maximum concentration close to 27 mg/L during the first rain. The benzalkonium concentration (sum of C12 and C14) stayed high (up to 1 mg/L) even 5 months after the treatment. Benzalkonium concentration measured in stormwaters was low (0.2 µg/L) but with contaminated suspended solids (up to 80 µg/g). © 2012 Elsevier Ltd. All rights reserved.