Although oncological treatments are improving, the prognosis of non-small-cell lung cancer (NSCLC) patients has not. Several biomarkers related to prognosis have been evaluated, and M30 and M65 have been reported to be higher in patients with NSCLC than in healthy people. In the current study, we evaluated the clinical importance of the change in serum M30 and M65 values after chemotherapy in patients with NSCLC. Serum M30 and M65 values were measured before and 48 h after chemotherapy in thirty-two patients with advanced NSCLC. The importance of the change in the levels of these markers after chemotherapy was analyzed by univariate analysis. The median serum M65 and M30 values increased significantly after chemotherapy (p < 0.001). The median M30 value after chemotherapy was an important prognostic factor for both overall survival (OS) (p = 0.002) and progression-free survival (PFS) (p = 0.002). Stage and histopathological type were significant both for PFS and OS. Multivariate analysis showed that the median M30 value after chemotherapy was the only independent prognostic factor for PFS (p = 0.04, HR 5.4) and OS (p = 0.02, HR 11.49). Our results indicated that both serum M30 and M65 values increased after chemotherapy in patients with advanced NSCLC, and an elevated serum M30 value was an independent prognostic factor for both PFS and OS.