Electron paramagnetic resonance (EPR) spectroscopy is an established technique to site-specifically monitor conformational changes of spin-labeled biomolecules. Emerging in-cell EPR approaches aiming to address spin-labeled proteins in their native environment still struggle to reach a broad applicability and to target physiologically relevant protein concentrations. Here, we present a comparative in vitro and in-cell double electron− electron resonance (DEER) study demonstrating that nanomolar protein concentrations are at reach to measure distances up to 4.5 nm between protein sites carrying commercial gadolinium spin labels.