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BACKGROUND It is unclear whether the third-generation intraoperative parathyroid hormone assay can improve surgical outcomes over second-generation assay in primary hyperparathyroidism. We aimed to compare the rate of decrease and diagnostic accuracy between the two assays after parathyroid adenoma excision. METHODS Consecutive patients undergoing parathyroidectomy with intraoperative parathyroid hormone were analyzed. Blood was drawn before and 10 minutes and 20 minutes after excision of the adenoma. The same blood sample was run simultaneously in the second-generation assays (Elecsys PTH STAT) and third-generation assays (Elecsys 1-84 PTH). Biochemical cure meant >50% intraoperative parathyroid hormone decrease at 10 minutes. Cure meant normocalcemia 6 months after operation. RESULTS Relative to the second-generation assay, the value of the intraoperative parathyroid hormone level was less in the third-generation assay before excision (P < .001), at 10 minutes (P < .001), and at 20 minutes (P < .001). The intraoperative parathyroid hormone rate of decrease and the proportion of normalized post-excision intraoperative parathyroid hormone were greater in the third-generation assay (P < .001), but the prediction accuracy appeared similar between the 2 (91.5% vs 91.0%). Patients with worse renal function (estimated glomerular filtration rate <80mL/min/1.73m2) had a slower intraoperative parathyroid hormone decrease in the second-generation but not in the third-generation assay. CONCLUSION Despite comparable accuracy between the two generations of assay, the third-generation assay might be better than the second-generation assay because of the more rapid decrease in the intraoperative parathyroid hormone and a greater percentage of normalized intraoperative parathyroid hormone, regardless of baseline renal function.