There are many methods for biotin determination, but the principle, detection time, and suitable applications of these methods are different. In recent years, microbiological assays, high-performance liquid chromatography (HPLC), gas chromatography (GC), and more accurate and efficient analysis methods such as ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) assays for biotin analysis have been reported. However, microbiological assay and HPLC are two methods that are mature and the basis for various applications in biotin analyses. This chapter details the various steps involved in both a microbial and HPLC detection methods for biotin.