Background: Accurate preoperative localisation of the parathyroid adenoma is essential to achieve a minimally invasive parathyroidectomy. The purpose of this study was to validate and improve our single-isotope dual-phase parathyroid imaging protocol utilising 99mTechnetium-Sestamibi ([99mTc]MIBI). There has been no accepted gold standard evidence-based protocol regarding timing of single-photon emission computed tomography/computed tomography (SPECT/CT) acquisition in parathyroid imaging with resultant variation between centres. We sought to determine the optimum timing of SPECT/CT post administration of [99mTc]MIBI in the identification of parathyroid adenomas. We aimed to evaluate the efficacy of early and late SPECT/CT and to establish whether SPECT/CT demonstrates increased sensitivity over planar imaging. Material and methods: A sample of 36 patients with primary hyperparathyroidism underwent planar and SPECT/CT acquisition 15 minutes (early) and two hours (late) post [99mTc]MIBI administration. Two radionuclide radiologists reviewed the images and Fisher’s exact Chi-squared statistic was used to evaluate the diagnostic performances of early versus late SPECT/CT acquisition and SPECT/CT versus planar imaging. Results: Twenty-one likely parathyroid adenomas were identified with a statistically superior diagnosis rate in the late SPECT/CT acquisition compared with both early SPECT/CT and planar imaging (p < 0.05). All adenomas diagnosed on early SPECT/CT acquisition were also identified on late SPECT/CT images. Conclusions: Single late phase SPECT/CT is significantly superior to early SPECT/CT in the identification of parathyroid adenomas. Late SPECT/CT improves diagnostic accuracy over planar acquisition. Imaging protocols should be revised to include late SPECT/CT acquisition. Early SPECT/CT acquisition can be eliminated from scan protocols with associated implications regarding reduced scan time and increased patient throughput.