Approximately 70% of medical decisions are made based on results of laboratory investigations. Immunochemical methods are used most commonly in routine laboratory diagnostics of endocrine disorders. Those methods are limited by susceptibility of the immunochemical reaction to various interferences. Interference may be caused by the presence of autologous antibodies, heterophilic antibodies, or paraproteins in the blood serum, by cross-reactions with similar reagents, haemolysis, significant lipidaemia, or hyperbilirubinaemia. Some recent reports have indicated a significant effect of biotin on the reliability of laboratory investigations. Biotin is a water-soluble vitamin belonging to the B group. It is present in popular dietary supplements — alone or as a component of multi-vitamin formulas — and it is advertised as a remedy to falling out and fragility of hair and nails. Due to its low molecular weight and a strong affinity to streptavidin, biotin is used in many immunochemical tests. Due to a strong and stable bond of streptavidin and biotin, analytical methods using the streptavidin (avidin)–biotin system are characterised by superior sensitivity, and they allow determination of very low levels of the tested substance in biological material. The presence of exogenous biotin in a sample may cause interference when using tests that utilise the streptavidin (avidin)–biotin system. Interference of biotin with immunochemical tests depends on several factors: the construction of the immunochemical test, the dose used by the patient, the biotin concentration in the sample, and most of all — the time from the last dose to the collection of biological material for laboratory testing. In this paper we present some practical recommendations and a procedure to be followed in the case of suspected interference of biotin in immunochemical assays, for clinicians and laboratory diagnosticians.