In addition to the basic nutrients (energy, protein, vitamins and minerals), plant tissues possess micro constituents that are essential for human well-being, productive and healthy lifestyle. They are known as bioactive, physiologically-active, functional or health-promoting substances and are found in commonly consumed fruits, vegetables and grains as secondary metabolites. Majority of these bioactive components (e.g., carotenoids, anthocyanins, phenolics) impart their physiological activities as antioxidants, and some of them such as several members of carotenoids have more than one mode of action, i.e., in addition to being antioxidants, they also possess vitamin A activity. Recent research has supported the role of carotenoids in maintaining human health and preventing or reducing the risk of chronic diseases. This has led to a growing interest in scientific communities to better understand their molecular and structural properties and their efficacy as health promoters. Certainly, this requires a collective effort to develop and validate methodologies and techniques that are capable to separate, quantify and identify such minor compounds and their metabolites as well. The present review was aimed to present the up-to-date analytical techniques used for the separation and quantification of carotenoids found in food and plasma. Additionally, their occurrence, physiological properties, extraction, purification and stability were discussed.