It is estimated that proteases comprise nearly 2% of the human genome. Given that the primary structure of all known proteases will soon be available, an important challenge is to define the structure-activity relationships that govern substrate hydrolysis. Ideally this would be accomplished on a genome-wide scale. To this end, we have developed a one-pot phage selection system that yields the substrate recognition profile of multiple proteases from a single round of selection. The system meets five key criteria: (i) multiple proteases can be analyzed simultaneously, (ii) prior knowledge of substrate preference is not required, (iii) information regarding substrate preferences on both side of the scissile bond is obtained, (iv) the system yields selective substrates that distinguish closely related proteases, and (v) semiquantitative information on substrate hydrolysis is obtained, allowing for the assignment of initial rank-order preferences. As an illustration, a phage selection with a mixture of thrombin and factor Xa (serine proteases) along with matrix-metalloproteinase-9 and atrolysin C (metalloproteinases) was performed. Peptide substrates were identified that (i) have high k(cat)/K(m) ratios, (ii) are selective for individual proteases, and (iii) match the sequences of known physiological substrates.