This article presents the development and validation of a new high performance liquid chromatography (HPLC) method coupled with diode array detector for the simultaneous analysis of benzalkonium chlorides (benzyl dimethyl dodecyl ammonium chloride, C12-BZCL, benzyl dimethyl tetradecyl ammonium chloride, C14-BZCL and benzyl dimethyl hexadecyl ammonium chloride, C16-BZCL) from wastewater samples. The chromatographic analysis was performed on an Acclaim Surfactant Plus (150 x 3.0 mm, 3 μm d.p.) column acquired from Thermo Scientific, kept at 30°C. All experiments were performed in isocratic elution conditions at a flow-rate of 0.5 mL/min. Mobile phase composition was a mixture of acetonitrile (A) and 0.2 M ammonium acetate solution (50:50, v/v). UV–VIS detection was λ = 262 nm for the three BZCL. The limits of detection (LODs) were obtained 15 µg/L (C12-BZCL), 19 µg/L (C14-BZCL) and 25 µg/L (C16-BZCL). The calibration curves were linear between 1 mg/L-100 mg/L, with R2 values above 0.999 for all BZCL. Solid phase extraction (SPE) using polymeric (Strata X) cartridges has been applied to extract and concentrate the target analytes from the wastewater. BZCL recoveries after SPE procedure were situated between 86.0÷94.6%. Intra-day and inter-day precision (RSD%) were situated between 1.4 ÷ 2.3% and 6.2 ÷ 6.7%, respectively. Limits of quantification (LOQs) was 4.5 µg/L (C12-BZCL), 5.6 µg/L (C14-BZCL) and 7.6 µg/L (C16-BZCL). © 2023, Politechnica University of Bucharest. All rights reserved.