Exclusive breastfeeding is recommended by the WHO for the first 6 mo of life because human milk protects against gastrointestinal infections and supplies balanced and adequate nutrient contents to the infant. However, reliable data on micronutrient concentrations in human milk are sparse, especially because some micronutrients are affected by maternal diet. Microbiological and competitive protein-binding assays, nuclear magnetic resonance or inductively coupled plasma spectroscopy, and chromatographic analyses are among the methods that have been applied to human-milk micronutrient analysis. However, the validation or evaluation of analytical methods in terms of their suitability for the complex human-milk matrix has been commonly ignored in reports, even though the human-milk matrix differs vastly from blood, plasma, or urine matrixes. Thus, information on the validity, accuracy, and sensitivity of the methods is essential for the estimation of infant and maternal intake requirements to support and maintain adequate milk micronutrient concentrations for healthy infant growth and development. In this review, we summarize current knowledge on methods used for analyzing water- and fat-soluble vitamins as well as iron, copper, zinc, iodine, and selenium in human milk and their different forms in milk; the tools available for quality control and assurance; and guidance for preanalytical considerations. Finally, we recommend preferred methodologic approaches for analysis of specific milk micronutrients.