This protocol outlines a general approach for characterizing the protospacer-adjacent motifs (PAMs) of Cas9 orthologs. It uses a three-plasmid system: One plasmid carries Cas9 and its tracrRNA, a second targeting vector contains the spacer and repeat, and the third plasmid encodes the targeted sequence (as the protospacer) with varying PAM sequences. It leverages the Cas9 nuclease activity to cleave and destroy plasmids that bear a compatible PAM. The level of depletion of a library of targeted plasmids after Cas9-mediated selection can then be assessed by deep sequencing to reveal candidate PAMs for downstream validation.