Resistance to chronic myeloid leukemia (CML) drug STI571 has been associated with point mutations in the kinase domain of BCR-ABL. For example, the mutation T315I (g. 68721C>T) and, to a lesser extent, Y253F (g. 58796A>T) appear in a significant proportion of patients resistant to treatment. Mutations appear intimately related to the development of resistance, and they may pre-exist in a small percentage (<1%) of tumor cells at the time of treatment initiation. Most mutation detection methods, including sequencing, are unable to detect such a small percentage of mutations in a background of wild type sequences. We describe the simplification and modification of a recently developed enhanced PCR-RFLP method, and its application to the detection of T315I and Y253F mutations. The method is quantitative, can be used in agarose gel or real time PCR formats, and reliably detects 1 mutation-containing cell in a background of almost 1000 non-mutated cells. The increased sensitivity offered by this assay will allow detection of these mutations at an early stage during treatment and will be useful in rational treatment modification and in studies which address the association between these mutations and drug resistance.