Insulinomas, although rare, cause considerable morbidity but are frequently amenable to surgical cure. Laparoscopic surgery can now be considered if the tumour is localised pre-operatively, but the optimal imaging approach has not been determined. The objective of this study was to evaluate the ability of different imaging investigations, including CT, MRI, endoscopic ultrasound, octreotide scintigraphy and arterial stimulation with simultaneous venous sampling (ASVS), to localise insulinomas. All patients with biochemically proven insulinoma at our institution underwent ASVS along with other imaging investigations as part of their routine investigation. The results of these investigations were compared with histological findings. Twenty-eight patients with biochemically proven insulinoma confirmed by histology were identified. Ultimately ASVS localised a lesion in all patients. Seventeen patients (61%) had laparoscopic surgery. Tumor-detection rates for other imaging investigations included 43.5% of cases using CT, 71% using MRI, 86% using endoscopic ultrasound and 33% using octreotide scintigraphy. In four patients, the ASVS was the only test to correctly localise the lesion. ASVS should be considered routinely before surgery to ensure accurate localisation of insulinomas.