Human milk oligosaccharides (HMOs) are known to positively influence infant health. Extensive variation exists in the levels, diversity, and complexity of oligosaccharides in the milk of a lactating mother. Until recently, limited availability of HMOs hampered their use in clinical applications. Most HMOs are unique to human milk, and have proven difficult and expensive to isolate and synthesize. Added to that, analysis of these complex glycans in milk samples requires state-of-the-art analytical instruments and associated technologies. The current review provides a critical overview of methods used in HMO analysis, and highlights the importance of understanding the factors which influence their composition and structural diversity. We also discuss recently employed strategies to overcome the availability of HMOs at industrial scale including microbial metabolic engineering and chemoenzymatic techniques. Finally, we examine how these recent advancements have opened up new avenues for future research and nutraceutical applications.