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Systemic chemotherapy is widely used for treatment of metastatic colorectal cancer (CRC). However, many CRC patients show poor response to chemotherapy and suffered from toxicities. Thus, prediction of chemotherapy response is very important in effective therapy. In the present study, we investigated whether chemotherapy response of CRC can be predicted by nano-assisted laser desorption/ionization time-of-flight (NALDI-TOF) analysis of low mass ions in CRC patients' sera. Various methods for extraction of low mass ions in serum have been tested for NALDI-TOF analysis, and among the protocols tested, low mass ions in hexane extraction was most comfortable. Profiling for low mass ions in hexane extracts from 16 CRC patients' sera was performed before chemotherapy, and responsiveness was defined after chemotherapy. Eight CRC patients were stable disease (SD), and other 8 patients showed partial response (PR). Interestingly, peak intensity of low mass ion with 305.85 m/z was significantly lower in SD patients, and discriminated from PR patients. In our preliminary study, we showed an example of profiling of low mass ion in sera for predicting chemotherapy response of CRC patient, and the results suggest that information of low mass ions in sera can be translate for the clinical application.