The intestinal microflora lives in intimate contact with its surrounding intestinal wall and the bacteria can exert beneficial or deleterious effects on the host, depending on whether they are classified as probiotics or as pathogens. The interaction is determined on one hand by characteristics of the microorganisms, and on the other hand by characteristics of the intestinal wall. Together they determine the health status of the intestine. This review describes parameters and techniques (with advantages and disadvantages) available for poultry to identify the characteristics of the intestinal health, as constituted by three components: immunity, integrity, and functionality. To investigate intestinal immunity, in situ detection of various cell populations of the immune system with specific monoclonal antibodies using immunocytochemical staining is a reliable, semi-quantitative method. In vitro assays to measure functional aspects of T lymphocytes, B lymphocytes, plasma cells, natural killer cells, macrophages, and phagocytes are applicable to intestinal wall tissue. For investigation of intestinal integrity, in situ detection of villous height and crypt depth and their ratio, and villus arrangement is still an easy, routine histological method. In addition, expression levels of specific molecules, such as E-cadherin, different growth factors, and trefoil factor, seem promising parameters. To investigate functionality of the intestine, the permeability can be measured as the rate of transport of tracer molecules across the epithelial surface. Furthermore, determination of the level of mucus secretion and its composition are a valuable tool. These parameters for immunity, integrity and functionality, or a combination thereof, are indispensable to investigate the influence of intestinal microorganisms on intestinal health.