Electron paramagnetic resonance (EPR) spectroscopy can be applied as an effective and non-invasive spectroscopic method for analyzing samples with unpaired electrons. EPR is suitable for the quantification of radical species, assessment of redox chemical reaction mechanisms in foods, evaluation of the antioxidant capacity of food, as well as for the analysis of food quality, stability, and shelf life. It can be employed for evaluating and monitoring the drug release processes, in vitro and in vivo. EPR can be employed for the direct detection of free radical metabolites, and the evaluation of drug release mechanisms from biodegradable polymers; it can be employed for analyzing the drug antioxidant effects. Additionally, spatial resolution can be achieved through EPR-imaging. EPR spectroscopy and imaging have shown diverse applications in food, biomedical and pharmaceutical fields, and also more applications are predictable to emerge in the future. This review highlights recent advances and important challenges related to the application of EPR in food, biomedical and pharmaceutical analysis and assessment.