Introduction: Vimentin has shown to be highly implicated in cancer initiation and progression. Vimentin is often a target of post-translational modifications (PTMs) which can be disease specific, thus targeting these specific modifications can be of high biomarker potential. In this study we set out to evaluate the biological relevance and serum biomarker potential of citrullinated vimentin (VICM) and non-citrullinated vimentin (VIM) in non-small cell lung cancer (NSCLC) and chronic obstructive pulmonary disease (COPD). Methods: A competitive ELISA targeting VIM was developed and quantified in serum from patients with NSCLC and COPD. VIM was compared with levels of VICM in the same indications. Results: VIM was significantly increased in NSCLC (n = 100) compared to healthy controls (n = 67) in two independent cohorts (p = 0.0003 and p < 0.0001). Furthermore, VIM was highly increased in late stages of NSCLC (p = 0.001), however VIM was not increased in COPD patients (n = 10). Contrarily, serum levels of VICM was not increased in late stages of NSCLC, but highly elevated in patients with COPD (p < 0.0001). Conclusions: These findings suggest a biomarker potential of VIM in NSCLC. Our findings also indicate that PTMs of vimentin are highly relevant and that targeting these modifications can have differential biomarker potential.