The effect of sample preparation on dentin electron paramagnetic resonance (EPR) spectra was investigated. The analysis was performed on dentin samples prepared by pure mechanical treatment or by an alkaline deproteination method. It was observed that (1) mechanical treatment induces both stable and transient signals, depending on the specific mechanical operation applied; and (2) sodium hydroxide deproteination removes the native signal, but at the same time introduces new confounding signals in the EPR spectrum. Also, it increases the radiation sensitivity of dentin. These findings suggest that attention must be paid to dentin sample preparation.