Cu is an essential trace element for Human. As free Cu is highly detrimental, the cell develops homeostasis mechanisms to control Cu load. However, Cu homeostasis can be affected in pathologies such as in cancer in which Cu use is optimized. This prompts the need to understand the mechanisms underlying these phenomena, and to further develop anti-Cu therapeutic strategies. We developed innovative Cu(I) ionophores and proved that they can kill cancer cells. Recent X-ray fluorescence microscopy experiments enabled us to show that these compounds disrupt intracellular Cu distribution. To deepen our understanding of both Cu homeostasis in cancer cells and the mechanism of action of these ionophores, we need to study Cu speciation in cancer cells in different conditions (exposed to Cu and/or Cu(I) ionophore) and compare it with healthy cells. Altogether, these data will reveal the evolution of the status of Cu upon cancer development and the impact of Cu(I) ionophore on this status.