The variability of human milk (HM) composition renders analysis of its components essential for optimal nutrition of preterm fed either with donor's or own mother's milk. To fulfil this requirement, various analytical instruments have been subjected to scientific and clinical evaluation. The objective of this study was to evaluate the suitability of a rapid method for the analysis of macronutrients in HM as compared with the analytical methods applied by cow's milk industry. Mature milk from 39 donors was analysed using an infrared human milk analyser (HMA) and compared with biochemical reference laboratory methods. The statistical analysis was based on the use of paired data tests. The use of an infrared HMA for the analysis of lipids, proteins and lactose in HM proved satisfactory as regards the rapidity, simplicity and the required sample volume. The instrument afforded good linearity and precision in application to all three nutrients. However, accuracy was not acceptable when compared with the reference methods, with overestimation of the lipid content and underestimation of the amount of proteins and lactose contents. The use of mid-infrared HMA might become the standard for rapid analysis of HM once standardisation and rigorous and systematic calibration is provided.