A non-radioactive method to determine 5-Fluorouracil (5FU) incorporation into DNA has been developed. Isolated DNA was enzymatically degraded to bases and the resulting 5FU was measured with standard gas-chromatography coupled to mass spectrometry (GC-MS) and compared with that of radioactive 5FU in a cell line. Incorporation into DNA of the murine Colon 26-B tumor treated with maximal tolerated doses of 5FU and fluorodeoxyuridine (FUdR) was maximal after 2 hour and was 15.4 and 71.0 fmol/microg DNA, respectively. After a plateau for about 3 days a decrease was observed to +/- 2 fmol/microg DNA after 10 days. The assay is very sensitive and reproducible and can be used in a clinical setting.