Pentachlorophenol (PCP) is a widespread and persistent hydrophobic organic pollutant in the environment despite its restricted public use. Risk assessment of such hydrophobic organic compounds (HOCs) is challenging because sorption and volatilization issues during toxicity test often lead to inconsistent exposure concentration. Considering the hydrophobicity of the PCP, in this study, a passive dosing format was applied by adopting a silicone O-ring as a reservoir and evaluated its applicability on the determination of PCP on Daphnia magna. Results obtained with passive dosing method were compared with that of solvent spiking method. We hypothesized that the passive dosing method may provide more reliable and accurate toxicity results than conventional solvent spiking approach. As a result, the partition coefficient of PCP between methanol and a test medium (log KMeOH:ISO) was 2.1, which enabled the maintenance of reliable exposure concentration throughout the experiment. In the acute toxicity tests, passive dosing and solvent spiking showed similar EC50 values of 576 and 485 µg/L for 24 h, and 362 and 374 µg/L for 48 h, respectively, which overlap with EC50 values of previous studies. Altogether, both methods were suitable for the acute toxicity assessment of hydrophobic PCP.