The purpose of this study was to optimize effective, but technically challenging99mTc-sestamibi/123I subtraction SPECT/CT protocol for parathyroid scintigraphy. An anthropomorphic parathyroid phantom was set up using a small sphere, a thyroid phantom and a thorax phantom with clinical range of activities of123I and99mTc. SPECT/CT acquisitions were performed using three collimators (Low Energy High Resolution (LEHR), Low Energy Ultra High Resolution (LEUHR) and Medium Energy Low Penetration (MELP)) and two energy window settings. Images were reconstructed with a combination of four different numbers of iterations and with or without scatter correction. Images were subjected to visual and quantitative evaluation. The effect of collimator, energy window selection and reconstruction parameters had a significant effect on visual appearance and adenoma contrast in parathyroid99mTc-sestamibi/123I subtraction SPECT/CT. Symmetrical energy windows and ultra-high resolution collimator yielded best results with some improvement with scatter correction.