Abstract Background Ionized calcium refers to the calcium ions that are present in extracellular fluid which facilitate a multitude of metabolic and physiological processes. As such, ionized calcium may be measured in patients with suspected abnormality in calcium metabolism, including a variety of bone diseases, chronic renal disease, and tetany. Ionized calcium is known to be unstable when exposed to air and many factors including specific handling procedures after specimen collection are known to affect measured analyte concentrations. The present study was conducted to assess the stability of serum ionized calcium in various storage conditions and evaluate stability of specimens when using the sample tray available on the Nova Prime ES Comp Plus analyzer. Methods Studies were performed to evaluate ambient (20–25 °C), refrigerated (2–8 °C), frozen (−20 °C) and freeze/thaw stabilities for ionized calcium using the Prime ES. Residual specimens stored refrigerated for up to 3 days were collected to create pools (n = 3–4). Each pool was then split into single-use aliquots to be stored and measured in the following conditions (temperature [timepoints]): Ambient (8, 24 h), Refrigerated (1, 2, 4 weeks), Frozen (1, 2, 4, 9 weeks), Freeze/Thaw (1, 2, 3 freeze/thaws). An aliquot of each pool was analyzed to obtain a baseline value. All aliquots were tested in duplicate, and the average was used to calculate % change for each pool and for all pools combined at each time point. Additionally, as ionized calcium is known to be unstable in specimens open and exposed to air, onboard specimen stability using the sample tray available on the analyzer was evaluated. Residual specimens used for previous stability studies were used to create pools (n = 4) on the same day. Each pool was split into sample cups (0.5 mL and 2.0 mL) and tested immediately using all the positions available on the sample tray (n = 10). For each pool, the first sample that was tested on the tray was used as a baseline value to calculate % change for the remaining cups on the tray. For all experiments, ionized calcium, pH, and pH-normalized ionized calcium results reported on the instrument were analyzed. Acceptable criteria was no greater than ±15% change from the baseline result. Results For the assessment of specimen stability at various storage conditions, the overall average % change of all pools combined at all time points ranged −3.9% to 2.1% for ionized calcium, −0.2% to 1.5% for pH and −1.4% to 5.9% for pH-normalized ionized calcium. For the sample tray stability evaluation, the % change in each sample tray position compared to baseline ranged −2.4% to 5.2% for ionized calcium, −0.1% to 1.6% for pH and 0.0%–3.9% for pH-normalized ionized calcium. It took up to 15 min to test all positions available on the sample tray. Conclusions Ionized calcium, pH and pH-normalized ionized calcium remained stable for 24 h ambient, 4 weeks refrigerated, 9 weeks frozen and up to 3 freeze/thaws. The sample tray available on the Prime ES was also found to be acceptable for use.