© 2022 American Chemical Society.The study of non-polar compounds in aqueous environments has always been challenging due to their poor solubility in aqueous media. The low affinity of non-polar compounds toward polar solutions facilitates their attachment to glassware, which results in unstable sample concentrations. To address this challenge, and to enable the preparation of a stable mixture of hydrophobic compounds in an aquatic environment, we introduce an in-vial standard water generating system consisting of a vial containing appropriate aqueous solution and a polydimethylsiloxane thin film spiked with target compounds. In this system, a solution with a stable analyte concentration is attained once equilibrium between the thin-film and aqueous solution has been achieved. The developed standard water system was studied using endocannabinoids and phospholipids as model hydrophobic compounds of biological importance, with results indicating that the concentration of hydrophobic compounds in water can remain stable over multiple days. The results also showed that analytes released from the thin film can compensate for analyte loss due to extractions with solid-phase microextraction fibers, thereby re-establishing equilibrium. Thus, the vial is suitable for the repeatable generation of non-polar standards for routine analysis and quality control. The results of this work show that the developed system is stable and reproducible and therefore appropriate for studies requiring the measurement of free concentrations and accurate quantification.