Three peripherally circulating hormones that can be measured in saliva have received growing attention in psychobiology research. Cortisol and dehydoepiandrosterone (DHEA) are steroid hormones indicative of activity in the hypothalamic–pitutary–adrenal (HPA) axis. The third, methoxyindole melatonin, is the hormonal product of the pineal neuroendocrine system. The development of reliable methods for salivary hormone assessment was a key turning point for psychobiology research, as it enabled new approaches to the study of a wide range of individual difference factors. These biological indices provide meaningful objective measures that can be analysed in parallel to self-reported variables (e.g. stress/well-being) as well as sociodemographic, developmental, psychological and health variables. Saliva is an easy-to-access biological fluid, collection of which is convenient and does not require trained personnel. Indeed, participants can be shown how to undertake self-collection of samples, which enables repeated sampling in ambulatory studies (with resultant ecological validity) as well as in relation to experimental manipulations within the laboratory. The purpose of this chapter is to guide the psychobiology researcher on appropriate approaches and methodologies for using salivary hormone measures for meaningful investigation of a virtually limitless range of potential research questions.