As compared to other classes of metabolites, determination of organic acids is an underrepresented field in cancer research and till now there has been a lack of appropriate analytical procedure for determination of serum levels of organic acids potentially associated with cancer development. The aim of the study was to develop a new rapid liquid chromatography–tandem mass spectrometry method for the quantification of six low-molecular-weight organic acids in human serum and to apply this method in an analysis of samples collected from non-small cell lung cancer (NSCLC) patients and a matched control group. The samples were prepared by solid phase extraction (Clean-up CUQAX, UCT). Chromatography was conducted on a Synergi Hydro–RP column (Phenomenex) and a gradient run of 15 min. Detection was performed using a negative multiple reaction monitoring mode. The calibration ranges were as follows: 0.24–38.42 μmol/L for 2-hydroxybutyric acid, 0.09–17.23 μmol/L for fumaric acid, 0.08–15.13 μmol/L for glutaric acid, 0.11–2.22 mmol/L for lactic acid, 0.39–30.98 μmol/L for pyroglutamic acid, and 0.08–16.93 μmol/L for succinic acid. Mean relative recovery range was 85.99–114.42% and the determined intra- and inter day coefficients of variation were ≤14%. Among the studied acids, pyroglutamic acid showed the best discriminating potential and enabled to identify accurately NSCLC patients and control subjects regardless of the cancer stage. Further investigations of serum organic acids may allow us to better understand the underlying mechanisms involved in NSCLC and develop novel means of its detection and treatment. The developed method may be also a valuable tool to study metabolic changes associated with other types of cancer.