Objective: Successful adrenal venous sampling (AVS) is traditionally assessed using the ratio of cortisol in adrenal to peripheral venous plasma to calculate the selectivity index. With mass spectrometry other steroids can be simultaneously measured that may improve numbers of apparent successful sampling procedures. Design: Cross-sectional multicenter study. Patients: The study involved 229 patients who underwent unstimulated AVS for subtyping primary aldosteronism. Measurements: Adrenal and peripheral venous plasma cortisol, 11-deoxycortisol, androstenedione and dehydroepiandrosterone were measured by mass spectrometry to assess AVS selectivity. Ratios of aldosterone, measured by mass spectrometry, to each of the four steroids in right versus left adrenal venous plasma were used to assess lateralisation. Results: Selectivity indices for 11-deoxycortisol, androstenedione and dehydroepiandrosterone were respectively 5.7–(CI 5.1–6.2), 5.1–(CI 4.8–5.4) and 5.9–(CI 5.5–6.4) fold higher (p < 0.0001) than for cortisol. At selectivity index cut-offs of ≥ 3 and ≥ 2, rates of bilateral successful AVS were respectively 91% and 93% for 11-deoxycortisol, 90% and 91% for androstenedione and 89% and 91% for dehydroepiandrosterone, all higher (p < 0.0001) than the 69% and 85% respective success rates for cortisol. With 11-deoxycortisol, rates of apparent unsuccessful AVS procedures were reduced by 54%–72% compared to use of cortisol. There were no clear advantages of any single steroid over the others to assess lateralized aldosterone secretion. Conclusions: 11-Deoxycortisol, androstenedione and dehydroepiandrosterone are more suitable biomarkers than cortisol to assess AVS selectivity and allow rescue of many procedures otherwise deemed unsuccessful. Measurements of cortisol to assess AVS selectivity are best replaced by mass spectrometric measurements of other steroids.