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Abstract The intricate nature of eukaryotic cells with intracellular compartments having differences in component concentration and viscosity in their lumen provides (membrane‐active) enzymes to trigger time‐ and concentration‐dependent processes in the intra‐/extracellular matrix. Herein, membrane‐active, enzyme‐loaded artificial organelles (AOs) are capitalized upon to develop fluidic and stable proteinaceous membrane‐based protocells. AOs in protocells induce the self‐assembly of oligopeptides into an artificial cytoskeleton that underlines their influence on the structure and functionality of protocells. A series of microscopical tools is used to validate the intracellular assembly and distribution of cytoskeleton, the changing protocells morphology, and AOs inclusion within cytoskeletal growth. Thus, the dynamics, diffusion, and viscosity of intracellular components in the presence of cytoskeleton are evaluated by fluorescence tools and enzymatic assay. Membrane‐active alkaline phosphatase in polymersomes as AOs fulfills the requirements of biomimetic eukaryotic cells to trigger intracellular environment, mobility, viscosity, diffusion, and enzymatic activity itself as well as high mechanical stability and high membrane fluidity of protocells. Thus membrane‐active AOs in protocells provide a variable reaction space in a changing intracellular environment and underline their regulatory role in the fabrication of complex protocell architectures and functions. This study contributes significantly to the effective biomimetics of cell‐like structures, shapes, and functions.