This research aimed to determine in vitro potential drug-drug interactions between anastrozole and commonly used analgesic drugs such as codeine and/or APAP, individually or in combination. Moreover, an additional goal in vivo was to clarify the potential impact of codeine and/or APAP on the metabolic pathway of anastrozole and, therefore, on anastrozole activity. Up to 80% of the diagnosed breast cancers express oestrogen receptors. Following their activation by oestrogens, oestrogen receptors induce proliferation of the tumour cells, therefore increasing cancer growth. Anastrozole is a third-generation aromatase inhibitor acting on the aromatase (CYP19A1), a key enzyme of the biosynthesis of oestrogens that is highly expressed by breast cancer cells. Paracetamol is the most used over-the-counter medication used either in single-compound form for mild pain or in multi-compound form as with an opioid for moderate to severe pain. Predominantly, paracetamol is combined with codeine. Codeine is a pro-drug, leading to morphine and its metabolites (morphine-3-glucuronide and morphine 6-glucuronide). Morphine acts on the mu-opioid receptors to produce analgesia. Mice livers were used to generate microsomes to determine the production of the anastrozole metabolite, hydroxy-anastrozole and impact of analgesic drugs on anastrozole metabolism in vitro using IC50 assay. For the in vivo experiments, female CB57BL/6J mice were treated with anastrozole (control condition) and codeine, paracetamol, or a combination of both in a single dose (acute treatment) or repeated injections over one week (chronic treatment). Both in vitro and blood samples were analysed and quantified by the liquid chromatography coupled to mass spectrometry (LCMS/ MS) using an isotope dilution method. In vitro, codeine and APAP showed an inhibitory effect on hydroxyanastrozole production in a dose-dependent matter. In vivo, all chronic treatments had a higher ratio of metabolite/parent drug than in the acute Page | 6 treatment, suggesting higher production of the hydroxy-anastrozole. Furthermore, differences between treatments of the same condition indicated that the combination of analgesic drugs and anastrozole had a lower metabolite/parent ratio, suggesting higher alternative metabolic pathways. These findings suggest that codeine and paracetamol impact anastrozole metabolism in mice and, therefore, could potentially impact the metabolism in humans. Further studies in humans are essential to assess the impact of potential interactions between breast cancer endocrine therapy and analgesic treatment with reducing prospective limitations.