Introduction: The regular diagnostic workup for primary aldosteronism (PA) can be very demanding and involves multiple invasive as well as time and cost intensive diagnostic tests. Here we have explored the value of urinary steroid metabolome analysis in the diagnosis and differential diagnosis of PA. Previously, urinary 3α,5β-tetrahydroaldosterone (THAldo) has been suggested as a reliable screening test for PA and serum 18-oxocortisol and 18-hydroxycortisol have been reported as diagnostic markers with the potential to distinguish unilateral aldosterone-producing adenomas (APA) from bilateral adrenal hyperplasia (BAH). Patients and methods: We studied 180 PA patients (103 APA, 71 BAH) in whom PA had been confirmed by saline infusion test followed by adrenal vein sampling for subtype differentiation. We carried out targeted sequencing for disease-causing somatic mutations in the KCNJ5, CACNA1D, ATP1A1 and ATP2B3 genes in tumour tissue obtained by unilateral adrenalectomy, which was available in 75/103 APA patients. The urine steroid metabolome was analysed by gas chromatography-mass spectrometry comprising the quantification of 38 distinct steroids including metabolites of aldosterone, deoxycorticosterone, corticosterone as well as 18OH-cortisol (18OH-F) and 18-oxo-tetrahydrocortisol (18oxo-THF). Results: As expected, urinary excretion of mineralocorticoids (p 69 μg/24h, with significantly higher THAldo in APAs compared to BAH (p=0.0082). Similarly, APAs had significantly higher excretion of 18OH-F (p=0.0171) and 18oxo-THF (p=0.0005). Genetic analysis of APA tissue revealed mutations in KCNJ5 (n=29), ATPases (n=12) and CACNA1D (n=6) while in 28 patients no known mutation was identified. Patients with KCNJ5 mutations had significantly increased excretion of both 18OH-F (p=0.0002) and 18oxo-THF (p<0.0001) as compared to the other mutation groups. Conclusion: Urine steroid metabolomics is a promising approach for the diagnosis and differential diagnosis of PA.