Saliva is gaining increasing attention as a bioanalytical sample matrix. Mostly because of the easy and noninvasive collection, it is not only beneficial in endocrinological and behavioral science, but also in pediatrics. Saliva also has the advantage of being the only body fluid which can be collected even during physical exercise, for example, during sportive activities, and there are physiological characteristics that make it superior to serum/plasma or urine for specific scientific questions. This review provides an insight into the physiology of saliva formation, explaining how certain compounds enter this bodily fluid, and gives advice for collection, storage and analytical methods. Finally, it presents a number of reliable and proven applications for saliva analysis from scientific fields including endocrinology, sports medicine, forensics and immunology.