OBJECTIVE Significant biological variation in macronutrient content of breast milk is an important barrier that needs to be overcome to meet nutritional needs of preterm infants. To analyze macronutrient content, commercial infrared milk analyzers have been proposed as efficient and practical tools in terms of efficiency and practicality. Since milk analyzers were originally developed for the dairy industry, they must be validated using a significant number of human milk samples that represent the broad range of variation in macronutrient content in preterm and term milk. Aim of this study was to validate two milk analyzers for breast milk analysis with reference methods and to determine an effective sample pretreatment. Current evidence for the influence of (i) aliquoting, (ii) storage time and (iii) temperature, and (iv) vessel wall adsorption on stability and availability of macronutrients in frozen breast milk is reviewed. METHODS Breast milk samples (n = 1188) were collected from 63 mothers of preterm and term infants. Milk analyzers: (A) Near-infrared milk analyzer (Unity SpectraStar, USA) and (B) Mid-infrared milk analyzer (Miris, Sweden) were compared to reference methods, e.g. ether extraction, elemental analysis, and UPLC-MS/MS for fat, protein, and lactose, respectively. RESULTS For fat analysis, (A) measured precisely but not accurately (y = 0.55x + 1.25, r(2) = 0.85), whereas (B) measured precisely and accurately (y = 0.93x + 0.18, r(2) = 0.86). For protein analysis, (A) was precise but not accurate (y = 0.55x + 0.54, r(2) = 0.67) while (B) was both precise and accurate (y = 0.78x + 0.05, r(2) = 0.73). For lactose analysis, both devices (A) and (B) showed two distinct concentration levels and measured therefore neither accurately nor precisely (y = 0.02x + 5.69, r(2) = 0.01 and y = -0.09x + 6.62, r(2) = 0.02 respectively). Macronutrient levels were unchanged in two independent samples of stored breast milk (-20 °C measured with IR; -80 °C measured with wet chemistry) over a period of 14 months. CONCLUSIONS Milk analyzers in the current configuration have the potential to be introduced in clinical routine to measure fat and protein content, but will need major adjustments.