This protocol describes the measurement of antimicrobial activity under reducing conditions against anaerobic bacteria of the human normal flora. The protocol is modified from the original description by Robert Lehrer et al. (1991), which is well established for the analysis of antimicrobial peptides with aerobic bacteria. To perform analysis for facultative and strict anaerobes we modified the assay by the addition of the reducing agent DTT to assay medium and performed incubation steps in an anaerobic jar. It was successfully used with analysis of Bifidobacteria, Lactobacilli, Bacteroides and Escherichia coli and might be adapted to other anaerobic bacteria. Several antimicrobial peptides have been tested with that system: while most of them showed less antimicrobial activity under reducing conditions, human beta defensin-1 was only active under these conditions against some of the mentioned strains.