Simultaneous determination of multiple biomarkers can improve the effectiveness and accuracy of cancer diagnosis. Cortisol, cortisone, and 4-methoxyphenylacetic acid (4-Me) are metabolic biomarker group with high specificity and sensitivity for the diagnosis of non-small cells lung cancer (NSCLC), and the development of their simultaneous determination method is desired. Herein, a simple, sensitive, and low-cost method involving pipette-tip solid-phase extraction (PT-SPE) using anion exchange adsorbent (MAX) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of three biomarkers (cortisol, cortisone, and 4-Me) in human urine. The sample (0.1 mL), adsorbent (1.5 mg) and organic reagent (3.5 mL) of MAX-PT-SPE are less consumed, and have the advantages of easy access to raw materials, simple assembly, convenient on-site instant extraction, low pollution, and low cost. The limits of detection of the three biomarkers were 0.006-0.024 ng mL-1, the recoveries of three spiked levels (2, 50, and 500 ng mL-1) were 91.0%-99.3%, with the relative standard deviations (RSDs) ≤ 5.9%. Finally, the MAX-PT-SPE-LC-MS/MS method achieved the quantitative analysis of cortisol, cortisone, and 4-Me in urine of different patients of NSCLC. This method is expected to be used in the non-invasive auxiliary diagnosis of NSCLC, and it provides a new strategy for multi-molecular diagnosis and multi-omics combined diagnosis.Copyright © 2023 Elsevier B.V. All rights reserved.