The cytochromes P450 (CYP450) represent a major enzyme family operating mostly in the first step of xenobiotic detoxification in aquatic organisms. The ability to measure these CYP450 enzymes' activities provides a crucial tool to understand organisms' response to chemical stressors. However, research on CYP450 activity measurement is still limited and has had variable success. In the present study, we optimize, compile, and compare existing scientific information and techniques for a series of CYP450 biomarkers (EROD, MROD, ECOD, APND, and ERND) used on Daphnia magna. Additionally, we explored these CYP450 biomarkers' activities through the first 5 days of life of daphnids, providing a link between their age and sensitivity to chemicals. In the experiment, daphnids were exposed to an organophosphate pesticide (diazinon) from birth to measure the molecular response of the detoxification process. Our results suggest EROD as the most applicable biomarker for organisms such as D. magna, with a higher organophosphate detoxification rate in daphnids that are 2 and 5 days old. Additionally, a larger body size allowed a more accurate EROD measurement; hence, we emphasize the use of 5-day-old daphnids when analyzing their detoxification response.