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This study aimed to evaluate the feasibility of the chemical method to analyze exhaled breath condensate (EBC) leukotriene B4 (LTB4) level in humans. High-performance liquid chromatography with a UV detector was applied to quantify the inflammatory biomarker. The LTB4 concentration in the concentrated pooled EBC samples was 1.19 ng/μL, and the average LTB4 concentration of each EBC sample was 15.38 ng/μL. This analytical technique was feasible to evaluate the levels of inflammatory mediators such as LTB4 in human EBCs without any complicated sample pretreatment processes.