Gastrointestinal mucosa forms a surface that interacts with many external factors. Beside the digestion and absorption of nutrients, it also acts as a barrier to allergens, pathogens, and toxins. Leaky gut syndrome is defined as a gut mucosal barrier dysfunction, which results in abnormally increased intestinal permeability. Research shows that leaky gut syndrome (LGS) has a pathogenetic relationship with a series of gastrointestinal and extra-intestinal disorders. This review discusses the current understanding of intestinal barrier composition and pathological contribution of LGS to various diseases. The major aim of this paper is to review different methods for diagnostics and evaluation of intestinal wall permeability, identifying their priorities and disadvantages.