Acute Myeloid Leukemia (AML) is a heterogeneous disease characterised by the abnormal proliferation of immature myeloid cells. Despite advancements in treatment, it is a particularly fatal blood cancer with a median survival rate of only 12 to 18 months. Protein post-translational modifications (PTMs) have been identified to play a vital role in almost every process within cells and are therefore important targets for the early detection and therapeutic treatment of cancer. The Bassermann laboratory focuses on specific PTMs called ubiquitin. The ubiquitin-proteasome system (UPS) is responsible for the degradation, turnover and function of proteins, therefore playing a crucial role in maintaining homeostasis and influencing various cellular pathways. The UPS is frequently altered in cancer and presents, therefore, as an interesting target to identify new vulnerabilities and, subsequently, treatment approaches in cancer and especially AML. One core component of the UPS are E3 ubiquitin ligases which are essential for substrate recognition and, therefore, ubiquitylation of proteins. There are more than 700 E3 ligases in the human genome of which many are still poorly characterised. Previous experiments identified the ubiquitin ligase FBXO41 as a potential vulnerability of AML cells. Since the function FBXO41 in AML has not been studied yet, the aim of this study was to generate tools for the investigation of its function. These can later be transduced into AML cells to analyse the effect of FBXO41 on proliferation, apoptosis, metabolism, and cell fitness. A knockout vector was generated and transduced into THP1 and Molm13 cells. A successful knockout was confirmed in THP1 via a western blot that showed decreased protein expression and in Molm13 via TIDE analysis. Additionally, an overexpression vector was generated and transfected into HEK293T cells. A successful overexpression was confirmed by western blot that showed increased protein expression compared to an empty vector. Moreover, a comparison of the two western blots showed again that FBXO41 is presumably vital for AML proliferation as it is endogenously expressed in cancerous (THP1 and Molm13) cells but not in non-cancerous (HEK293T) cells. Taken together, within this study, we generated and validated tools to knockout and overexpress FBXO41 to further investigate its role in AML in order to subsequently identify new treatment approaches in AML.