The feasibility of using near-infrared (NIR) spectroscopy in combination with partial least-squares (PLS) regression was explored to measure electrolyte concentration in whole blood samples. Spectra were collected from diluted blood samples containing randomized, clinically relevant concentrations of Na+, K+, and Ca2+. Sodium was also studied in lysed blood. Reference measurements were made from the same samples using a standard clinical chemistry instrument. Partial least squares (PLS) was used to develop calibration models for each ion with acceptable results (Na+, R-2 = 0.86, CVSEP = 9.5 mmol/L; K+, R-2 = 0.54, CVSEP = 1.4 mmol/L; Ca2+, R-2 = 0.56, CVSEP = 0.18 mmol/L). Slightly improved results were obtained using a narrower wavelength region (470-925 nm) where hemoglobin, but not water, absorbed indicating that ionic interaction with hemoglobin is as effective as water in causing measurable spectral variation. Good models were also achieved for sodium in lysed blood, illustrating that cell swelling, which is correlated with sodium concentration, is not required for calibration model development.