Sodium (Na), potassium (K), and the ratio Na:K in human milk (HM) may be useful biomarkers to indicate secretory activation or inflammation in the breast. Previously, these elements have been measured in a laboratory setting requiring expensive equipment and relatively large amounts of HM. The aim of this study was to compare measurements of Na and K in HM using inductively coupled plasma optical emission spectrometry (ICP-OES) with small portable ion selective electrode probes for Na and K. Sixty-five lactating women donated 5 mL samples of HM. Samples were analyzed with two ion selective probes (Na and K) and also ICP-OES. The data were analyzed using paired t-test and Bland–Altman plots. Na concentrations were not significantly different when measured with ion selective electrode (6.18 ± 2.47mM; range: 3.59–19.8) and ICP-OES (5.91 ± 3.37 mM; range: 2.59–21.5) (p = 0.20). K concentrations measured using the ion selective electrode (11.7 ± 2.21 mM: range: 7.69–18.1) and ICP-OES (11.1 ± 1.55 mM: range: 7.91–15.2) were significantly different (p = 0.01). However, the mean differences of 0.65 mM would not be clinically relevant when testing at point of care. Compared to ICP-OES, ion selective electrode is sufficiently accurate to detect changes in concentrations of Na and K in HM associated with secretory activation and inflammation in the mammary gland.