HIV-1 protease is a key enzyme in the life cycle of HIV/AIDS, as it is responsible for the formation of the mature virus particle. We demonstrate here that phage-display peptides raised against this enzyme can be used as peptide sensors for the detection of HIV-1 protease in a simple, one-pot assay. The presence of the enzyme is detected through an energy transfer between two peptide sensors when simultaneously complexed with the target protein. The multivalent nature of this assay increases the specificity of the detection by requiring all molecules to be interacting in order for there to be a FRET signal. We also perform molecular dynamics simulations to explore the interaction between the protease and the peptides in order to guide the design of these peptide sensors and to understand the mechanisms which cause these simultaneous binding events. This approach aims to facilitate the development of new assays for enzymes that are not dependent on the cleavage of a substrate and do not require multiple washing steps.