Calcium ions are crucial in numerous physiological processes, and their precise measurement is important for many clinical diagnostics and therapeutic interventions. Traditional detection methods, such as atomic absorption spectroscopy, cannot meet clinical requirements, as they measure total calcium instead of the clinically relevant ionized form (Ca²⁺ ). Ion-selective electrodes (ISEs) provide a convenient, accurate, and specific method for Ca²⁺ determination. Here, two isomeric calcium ionophores (R ,R )-calcium ionophore I, also known as ETH 1001, and (R ,S )-calcium ionophore I are synthesized and characterized for the analysis of whole blood samples with the Eaglenos blood gas analyzer (model: EG-i30) equipped with test cartridges containing screen-printed electrodes. (R ,R )-Calcium ionophore I demonstrated excellent precision in whole blood samples, achieving an average bias of -2.2% compared with the available gold standard. On the other hand, the (R ,S ) isomer was not satisfactory, exhibiting a bias of up to -20%. Ion transfer voltammetry at thin membrane films gave information about the complex stoichiometry, complex formation constants, and ion selectivity for the two isomeric ionophores. A stoichiometry of the Ca²⁺ -ionophore complex was confirmed to be 1:2 for both ionophores, while the (R ,R ) isomer gave 3.4 orders of magnitude larger complex formation constants and a modestly higher selectivity. While these data are valuable, the poor performance of membranes containing the (R ,S ) isomer is not directly apparent from the fundamental binding characteristics. It may be caused by interference from lipophilic blood sample components and/or surface adsorption processes, suggesting that routine selectivity characterizations of membranes containing selective ionophores are insufficient to assess their usefulness in clinical applications.