The aim of this study was to use calculated aldosterone concentration in the right adrenal vein (RAV) (cAldoRAV) for identifying the dominant side of aldosterone secretion in patients with primary aldosteronism undergoing adrenocorticotropic hormone-stimulated adrenal venous sampling (AVS). Patients with primary aldosteronism who had successful AVS (selectivity index > 3) were studied. Based on the assumption that cortisol production from both adrenal glands is equal, aldosterone concentration in the RAV was calculated by using the data from left adrenal vein (LAV) and inferior vena cava. The aldosterone concentration in the LAV (AldoLAV) compared to the cAldoRAV (AldoLAV:cAldoRAV ratio) was then used to determine the dominant side of aldosterone secretion compared with standard AVS interpretation using lateralization index (LI). The LI ≥ 4 indicated unilateral disease and LI ≤ 3 indicated bilateral disease. The LI between 3-4 was diagnosed as indeterminate. Sixty-eight patients with concordant results between AVS and adrenal imaging study (32 were left-sided, 22 were right-sided and 14 were bilateral) were selected for studying diagnostic performance. The AldoLAV:cAldoRAV ratio with the cutoff values of ≥ 3 and ≤ 0.33 could identify unilateral diseases (left-sided and right-sided disease, respectively) with 92.6% sensitivity and 100% specificity. The AldoLAV:cAldoRAV ratio can determine the dominant side of aldosterone secretion with high sensitivity and specificity when compared with standard AVS interpretation. It provides an option for identification of unilateral and bilateral disease in select patients if repeat AVS cannot be performed.