The cell’s interior contains a high concentration of macromolecules, which result in a highly crowded environment that affects the functionality of biomolecules due to excluded volume effects. These effects are often neglected in in vitro assays. Previous studies used poly ethylene glycol (PEG) based crowding sensors and artificial macromolecular crowding agents to investigate the crowding effects in vitro and were able to experimentally demonstrate a compression of the sensor as a result of a decreased accessible molecular volume. However, it is still unclear how the crowded environment affects the exact conformation of the sensor which precludes comparison with the molecular dynamics simulation results. We have developed a macromolecular crowding sensor based on a nitroxide spin-labelled 10okDa PEG polymer to probe the crowding effect in vitro using Electron Paramagnetic Resonance (EPR) techniques. In particular, we compared the crowding effects of large soluble polymers 20okDa PEG and 70okDa Ficoll with small molecular weight Sucrose. Radical relaxation times as probed by pulsed EPR showed that they are invariant with the nature of the crowding agent and therefore are not the result of excluded volume effects. Preliminary results of inter-nitroxide distance measurements using Double Electron-Electron Resonance (DEER) suggest that this method is capable of detecting conformational changes depending on the sensor’s concentration and nature of crowding agent. We are currently working on the synthesis of smaller molecular weight PEG polymer with shorter inter-spin distances, which should facilitate improved DEER measurements.