Botulinum neurotoxins (BoNTs) are bacterial proteins produced by Clostridium species causing lethal disease botulism. The widely used test for the identification of BoNTs in both clinical specimens and food is the mouse bioassay, which suffers by several limitations. Thus there is a need for a rapid and sensitive method for detecting of these BoNTs. Matrix assisted laser desorption (MALDI) mass spectrometry in combination with in vitro enzymatic assay Endopep-MS was recently demonstrated as a robust and fast technique for detection of BoNTs. In this study, we follow up this idea and use specifically designed peptide substrate biotinylated at both termini as a target for BoNT A. The products of the enzymatic reaction are peptide fragments of the original substrate that are detectable by MALDI mass spectrometry. We used MALDI chips functionalized with biotin-binding proteins streptavidin, neutravidin and avidin to enrich the biotinylated peptide fragments from crude biological matrices. These chips were prepared by modification of indium tin oxide glass using ambient ion soft landing under atmospheric pressure. One microliter of the sample after the specific BoNT A enzymatic reaction was applied on the MALDI chip. After incubation and washing the whole MALDI chips in buffer, each spot was covered by CHCA matrix. The resulting peptide fragments were measured by Autoflex MALDI mass spectrometer. The functionalized MALDI chips achieved low nonspecific interactions and efficient peptides ionization to detect BoNT A in samples. Two peptides, products of the enzymatic reaction, were observed in the spectra. The limit of detection for enriched peptides was 0.01 ng/ml of BoNT A concentration. The results indicate that detection of BoNT A using functionalized MALDI chips is sensitive, robust, fast and might be automated for general use in MALDI Biotyper system equipped laboratories.