Controlling infectious diseases is vital for poultry health and diagnostic methods are an indispensable feature to resolve disease etiologies and the impact of infectious agents on the host. Although the basic principles of disease diagnostics have not changed, the spectrum of poultry diseases constantly expanded, with the identification of new pathogens and improved knowledge on epidemiology and disease pathogenesis. In parallel, new technologies have been devised to identify and characterize infectious agents, but classical methods remain crucial, especially the isolation of pathogens and their further characterization in functional assays and studies. This review aims to highlight certain aspects of diagnosing infectious poultry pathogens, from the farm via the diagnostic laboratory and back, in order to close the circle. By this, the current knowledge will be summarized and future developments will be discussed in the context of applied state-of-the-art techniques. Overall, a common challenge is the increasing demand for infrastructure, skills and expertise. Divided into separate chapters, reflecting different disciplines, daily work implies the need to closely link technologies and human expertise in order to improve bird health, the production economy and to implement future intervention strategies for disease prevention.