With its inclusion under Action 3 in the Environment and Health Action Plan 2004–2010 of the European Commission, human biomonitoring is currently receiving an increasing amount of attention from the scientific community as a tool to better quantify human exposure to, and health effects of, environmental stressors. Despite the policy support, however, there are still several issues that restrict the routine application of human biomonitoring data in environmental health impact assessment. One of the main issues is the obvious need to routinely collect human samples for large-scale surveys. Particularly the collection of invasive samples from susceptible populations may suffer from ethical and practical limitations. Children, pregnant women, elderly, or chronically-ill people are among those that would benefit the most from non-invasive, repeated or routine sampling. Therefore, the use of non-invasively collected matrices for human biomonitoring should be promoted as an ethically appropriate, cost-efficient and toxicologically relevant alternative for many biomarkers that are currently determined in invasively collected matrices. This review illustrates that several non-invasively collected matrices are widely used that can be an valuable addition to, or alternative for, invasively collected matrices such as peripheral blood sampling. Moreover, a well-informed choice of matrix can provide an added value for human biomonitoring, as different non-invasively collected matrices can offer opportunities to study additional aspects of exposure to and effects from environmental contaminants, such as repeated sampling, historical overview of exposure, mother-child transfer of substances, or monitoring of substances with short biological half-lives.