Aquaculture, a mass supplier of seafood, relies on plastic materials that may contain the endocrine disruptors bisphenol-A (BPA) and tert-octylphenol (t-OCT). These pollutants present toxicity to Artemia, the live aquaculture feed, and are transferred through it to the larval stages of the cultured organisms. The purpose of this work is the development and validation of an analytical method to determine BPA and t-OCT in Artemia and their culture medium, using n-octylphenol as the internal standard. Extraction of the samples was performed with Η2Ο/ TFA (0.08%)–methanol (3:1), followed by SPE. Analysis was performed in a Nucleosil column with mobile phases A (95:5, v/v, 0.1% TFA in H2O: CH3CN) and B (5:95, v/v, 0.08% TFA in H2O: CH3CN). Calibration curves were constructed in the range of concentrations expected following a 24 h administration of BPA (10 μg/mL) or t-OCT (0.5 μg/mL), below their respective LC50. At the end of exposure to the pollutants, their total levels appeared reduced by about 32% for BPA and 35% for t-OCT, and this reduction could not be accounted for by photodegradation (9–19%). The developed method was validated in terms of linearity, accuracy, and precision, demonstrating the uptake of BPA and t-OCT in Artemia.