BACKGROUND: Biotin interference in biotin-streptavidin-based immunoassays is increasingly reported due to individuals taking biotin containing supplements and patients prescribed biotin. The reported prevalence of serum biotin threshold (> 10 µg/L) for interference in Roche Diagnostics immunoassay tests is 0.8% in Australia and 7.4% in the USA. There are, however, no such data in UK populations. In a service evaluation, we therefore studied the prevalence of biotin interference in routine serum samples received in our laboratory. METHODS: Biotin was measured in 524 anonymised surplus serum samples in which at least one immunoassay test had been requested. RESULTS: The median (95% confidence intervals) for serum biotin was 0.27 µg/L (95% confidence limits: 0.07-0.93 µg/L). Serum biotin was <10 µg/L in all samples, <5 ug/L in 522 (99.6%) and <1 µg/L in 513 (98.1%) samples. In 4 samples serum biotin was >2.5 µg/L (0.8%). CONCLUSIONS: These data indicate that the probability of biotin immunoassay interference in our patient population is extremely low, with the exception of assays reporting the lowest interference thresholds (e.g. Ortho Troponin I assay; threshold >2.5 µg/L).