A mid-infrared human milk analyzer (HMA) is designed to measure the macronutrients in human milk over a wide range of concentrations. Human milk samples (N = 30, 4 different dilutions each) were used to compare the macronutrient levels determined by the HMA to those derived from traditional laboratory methods. There was a small but statistically significant difference in the levels of fat, protein, lactose, total solids, and energy for all samples. These differences were consistent with subtle differences in the chemical principles governing the assays. For higher macronutrient levels, a trend to greater differences between the HMA and the laboratory method was seen, particularly in samples with high fat concentration. The intra-assay variation for the HMA for all macronutrients was less than 4%. It is concluded that that with appropriate sample preparation, the mid-infrared HMA can provide a practical measurement of macronutrients in human milk.