Metabolomes are small molecule metabolites (<1000 Da) produced by cellular processes. Metabolomes are close counterparts to the genome, transcriptome and proteome. The aim of this study was to develop a method to detect and quantify candidate nucleoside metabolomes 1-methyl adenosine (1-MA), 1-methylguanosine (1-MG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the urine of patients with breast cancer using gas chromatography-mass spectrometry (GC-MS). The method was applied to urine specimens from patients with breast cancer (n = 56) and benign breast tumors (n = 22), as well as from healthy females (n = 20). The relative standard deviations of precision and repeatability analysis were <10%, and recoveries ranged from 88.5 to 105.6%. Limits of detection were 0.014, 0.012, and 0.018 mg/L for 1-MA, 1-MG and 8-OHdG, respectively. The lower limits of quantitation were 0.056, 0.048 and 0.072 mg/L, respectively. There were significant differences in concentrations of candidate metabolomes between patients with cancer and the healthy individuals, especially for those in the early stages of the disease (p < 0.001). No significant differences were observed between the benign and healthy groups. In conclusion, a reliable GC-MS method for the detection and quantification of 1-MA, 1-MG, and 8-OHdG metabolomes in urine has been developed.