Ionizing radiation produces many kinds of free radicals in biologically important molecules through direct or indirect actions. In this chapter we have shown that a method that combined ESR, spin trapping and chromatographic separation made it possible to identify free radicals induced by direct ionization as well as OH-radical reactions in nucleic-acid related and protein-related compounds. In addition, combination with enzymatic digestion expanded the potential of this method to detect free radical production in molecules with high molecular weight such as polynucleotides, RNA and DNA, and polypeptides and proteins. The effects of ionizing radiation on biological molecules underlie the primary processes involved in radiation biology. ESR combined with spin trapping remains a useful method for clarifying these processes in a living organism.