Biotinylated molecules are of great interest in biotechnology and therefore used considerably. A big amount is established to allow the quantification of biotin derivatives. But because most of the previous described methods are rather insensitive and hence require a great amount of sample, a new assay for the quantification of biotinylated molecules is established, which consists of high sensitivity. The basic principle depends on the strong binding kinetics between avidin and biotin, as well as the use of a commercially available biotinylated fluorescent dye (biotin-4-fluorescein, B4F) for readout of the titration. After an initial adjustment and standardization the required solutions can be stored for months at -25C and -80C respectively. This procedure allows the quantification of the amount of biotin of an unknown sample in nanomolar range, only by a single titration. For verification of the new established assay a previous described method for quantification of the degree of biotinylation is used. The enhanced assay within the scope of the masters thesis is successfully tested on small biotin derivatives. In case of bigger biotinylated molecules, e.g. biotinylated DNA, systemic deviation can occur, which is caused by steric hindrance. This problem is eliminated by digestion with exonuclease I. Furthermore the applicability of the assay is tested for biotinylated proteins. Biotinylated proteins are indispensable tools in bioanalytics, medical diagnostics and in biotechnology. Anyway, in literature the amount of methods for characterization of the degree of biotinylation of protein is sparse [Rao et al., 1997]. The amount of biotin groups per protein has a significant impact on the functionality of the protein in the designated application [Narang et al., 1997], for this reason a practical and reliable method is required. To avoid false negative results due to steric hindrance, an ideal condition of digestion has to be found. In the present study the new established B4F-assay and the classic ANS-Assay were conducted in parallel to identify the ideal condition of digestion of biotinylated proteins.