Backgrounds: Primary aldosteronism (PA) is diagnosed and treated by the long steps, such as screening, confirmation testing and subtype diagnosis (computed tomography (CT) scan and adrenal venous sampling (AVS)). Objective: The aim of the study was to determine the role of peripheral plasma levels of 18-oxo-cortisol (p18oxoF) and 18-hydroxycortisol (p18OHF) in discriminating aldosterone producing adenoma (APA), which is a major surgically curable subtype of PA, from bilateral hyperaldosteronism (BHA) or primary hypertension (PH) which should be medically treated. Patients: The study included 79 PH patients with the aldosterone/renin activity ratio (ARR) of 9.0 ± 0.5 (ng/dl per ng/ml/h) and 237 PA patients, such as 87 patients with CT-positive macro APA (ARR: 363 ± 38.7), 29 patients with CT-negative micro APA (ARR: 64.8 ± 11.1) and 121 BHA patients (ARR: 87.1 ± 4.5). Methods: All PA patients underwent AVS, and all APA were surgically proven and histopathologically confirmed including immunohistochemical analysis of steroidogenic enzymes. We measured p18oxoF and p18OHF of all patients by high sensitive LC ms/ms. Results: APA patients showed significantly higher levels of p18oxoF and p18OHF compared to BHA or PH patients. Especially, the ROC analysis of 18oxoF in macro APA versus BHA or PH demonstrated clinically significant discrimination with higher sensitivity of 0.81 and higher specificity of 0.97 by cutoff value of 4.6 ng/dl. Conclusions: Peripheral plasma levels of 18-oxo-cortisol might practically differentiate aldosterone producing adenoma from bilateral hyperplasia and primary hypertension omitting many steps of diagnostic workup for PA.