Sortases are enzymes that are responsible for the attachment of secreted proteins to the cell wall of Gram-positive bacteria. Hereby, the sortases recognize short, five-residue amino acid sequences present in the target proteins and fuse them to the peptidoglycan layer via a transpeptidation reaction, creating a new peptide bond between the C-terminus of the recognition sequence and the cell wall. The transpeptidation activity of sortases is widely used in protein engineering for modification of target proteins. The majority of protocols rely on the high activity of the well-characterized Staphylococcus aureus SrtA and variants thereof, while sortases from other classes are not used for this purpose. This can be attributed to the lower activity of other sortases and to the limited sequence specificity data available for the different sortases. We set out to determine the sequence specificity of Bacillus anthracis SrtB. To this end, we developed a new method for sequence specificity determination of sortases or other bond-forming enzymes that recognize an amino acid sequence. Using mixtures of recognition peptides of limited complexity, which are reacted with biotinylated substrates, the biotinylated transpeptidation products are isolated with magnetic streptavidin beads and analyzed via liquid chromatography and mass spectrometry. With this, peptide sequences that are recognized by the sortase and function as substrates can be determined and quantified. The method, developed with the highly active evolved SrtA from S. aureus, allowed for the first time unbiased in-depth analysis of the sequence specificity for SrtB from B. anthracis, which is 104-fold less active than SrtA from S. aureus.