Humans and other living organisms are exposed to a variety of chemical pollutants that are released into the environment as a consequence of anthropogenic activities. Environmental pollutants are incorporated into the organism by different routes and can then be stored and distributed in different tissues, which leads to an internal concentration that can induce different alterations, adverse effects and/or diseases. Control measures should be taken to avoid these effects and human biomonitoring is a very useful tool that can contribute to this aim. Human biomonitoring uses different matrices to measure the target chemicals depending on the chemical, the amount of matrix necessary for the analysis and the detection limit (LOD) of the analytical technique. Blood is the ideal matrix for most chemicals due to its contact with the whole organism and its equilibrium with organs and tissues where chemicals are stored. However, it has an important disadvantage of being an invasive matrix. The development of new methodology and modern analytical techniques has allowed the use of other matrices that are less or non-invasive, such as saliva, urine, meconium, nails, hair, and semen or breast milk. The presence of a chemical in these matrices reflects an exposure, but correlations between levels in non-invasive matrices and blood must be established to ensure that these levels are related to the total body burden. The development of new biomarkers that are measurable in these matrices will improve non-invasive biomonitoring. This paper reviews studies that measure Cd, Pb, Hg, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides and phthalates in non-invasive matrices, the most used techniques for measurements and what alternative techniques are available.