We present a simple and inexpensive 'one-step' protocol for the hydrolysis of DNA to deoxyribonucleosides. Unlike the older DNA hydrolysis protocol which is cumbersome and labor intensive, this new protocol is ideal for high-throughput assays and is suitable automation. Using this protocol we were able to hydrolyze several hundred samples within an 8-hour period. The new protocol is fully compatible with LC-MS/MS and gives similar recoveries for all five major deoxyribonucleosides when compared to the older protocol.