Abstract: Chemical disequilibrium quantified using the available free energy has previously been proposed as a potential biosignature. However, researchers remotely sensing exoplanet biosignatures have not yet investigated how observational uncertainties impact the ability to infer a life-generated available free energy. We pair an atmospheric retrieval tool to a thermodynamics model to assess the detectability of chemical disequilibrium signatures of Earth-like exoplanets, focusing on the Proterozoic eon when the atmospheric abundances of oxygen–methane disequilibrium pairs may have been relatively high. Retrieval model studies applied across a range of gas abundances revealed that order-of-magnitude constraints on the disequilibrium energy are achieved with simulated reflected-light observations for the high-abundance scenario and high signal-to-noise ratios (50), whereas weak constraints are found for moderate signal-to-noise ratios (20–30) and medium- to low-abundance cases. Furthermore, the disequilibrium-energy constraints are improved by using the modest thermal information encoded in water vapour opacities at optical and near-infrared wavelengths. These results highlight how remotely detecting chemical disequilibrium biosignatures can be a useful and metabolism-agnostic approach to biosignature detection.