Background Identification of diseased and normal parathyroid glands during parathyroid surgery can be challenging. The aim of this study was to assess whether near-infrared (NIR) fluorescence imaging using administration of a low-dose Methylene Blue (MB) at the start of the operation could provide optical guidance during parathyroid surgery and assist in the detection of parathyroid adenomas. Methods Patients diagnosed with primary hyperparathyroidism planned for parathyroidectomy were included. Patients received 0.5 mg/kg MB intravenously directly after start of anesthesia. During the operation, NIR fluorescence imaging was performed to identify parathyroid adenomas. Imaging results were compared with a previous published feasibility study in which 12 patients received MB after intraoperative identification of the adenoma. Results A total of 13 patients were included in the current study. In 10 of 12 patients with a histologically proven adenoma, the adenoma was fluorescent. Mean signal to background ratio was 3.1 ± 2.8. Mean diameter of the resected lesions was 17 ± 9 mm (range 5–28 mm). Adenomas could be identified up to 145 minutes after administration, which was the longest timespan until resection. Interestingly, in 3 patients, a total of 6 normal parathyroid glands (median diameter 2.5 mm) with a signal to background ratio of 1.8 ± 0.4 were identified using NIR fluorescence imaging. Conclusion Early administration of low-dose MB provided guidance during parathyroidectomy by identifying both parathyroid adenomas and normal parathyroid glands. In patients in whom difficult identification of the parathyroid adenoma is expected or when normal glands have to be identified, the administration of MB may improve surgical outcome.