Although a range of polymer–protein polyion complex (PIC) micelle systems have been developed in the literature, relatively little attention has been paid to the influence of polymer structure on the assembly, or to the mechanism of disassembly. In this work, Förster resonance energy transfer is used in combination with light sheet fluorescence microscopy and isothermal calorimetry to monitor the formation and stability of PIC micelles with various carboxylic‐acid‐based binding blocks in MCF‐7 cancer spheroid models. All micelles are stable in the presence of free protein, but are unstable in solutions with an ionic strength >200 mm and prone to disassembly at reduced pH. Introducing carbon spacers between the backbone and the binding carboxylic acid results in improved PIC micelle stability at physiological pH, but also increases the pKa of the binding moiety, resulting in improved protein release upon cell uptake. These results give important insights into how to tune PIC micelle stability for controlled protein release in biological environments. Proteins suffer from low stability and often for biomedical applications unfavorable surface charge. Protecting proteins using polyion complex micelles can be a fast and efficient way to allow better transport into 3D cancer spheroids. In this work, the effect of a hydrophobic flexible spacer between polymer and the negative charge on the stability of the complex is investigated .