Human milk is the optimal nutrition for infants. Human milk not only provides abundant nutrients, it also contains non-nutritional bioactive components, like human milk oligosaccharides (hMOS), sialic acids (Sia) and corticosteroids, which have a short- and longterm effects on infant development. In order to better understand the complex functions of bioactive factors, it is increasingly important to perform quantitative analysis of specific components. However, reliable data on the bioactive factor content in human milk are sparse and inconsistent. Little is known regarding whether and how lactation and other factors contribute to the bioactive factor variability. Therefore, quantifying the concentrations of bioactive factors with reliable analytical techniques is needed. Subsequently, an assessment of the relationships between potential influencing factors and bioactive molecule concentrations can be made. In this thesis, we explored the concentrations of hMOS, Sia and corticosteroids in milk (human milk, infant formula, mouse milk) with state-of-the-art analytical techniques, and assessed the relationships between potential influencing factors and bioactive molecule concentrations. Our data contribute to better understand the complex functions of bioactive factors. Understanding the amounts of bioactive factors in human milk and how they fluctuate with lactation and other factors also bring new insights for promoting best breastfeeding practices.