The application of UV diode array detection in high-performance liquid chromatographic (HPLC) identification and quantitation of several classes of synthetic and commercially available alkylated nucleobases is investigated. Quantitative spectral overlays of these compounds to methyl standard references from a spectral library and absorbance ratios at two maximal wavelengths (λ(max)) are found to be useful in categorizing the solutes. They can be grouped into classes of compounds originating from a specific nucleobase and classes of analogs having different alkyl substituents (e.g., methyl, ethyl, propyl, allyl, and benzyl) at the same position of the heterocycle. At a selected wavelength for alkylated nucleobases in the same class, the detector response factors are independent of the alkyl group (±10%). This technique provides a practical means for both qualitative and quantitative analysis of product distribution of DNA base alkylation by using only readily obtainable methylated derivatives as the reference standards.