Peritoneal metastases are usually disseminated throughout the abdominal cavity and therefore difficult to see during surgery. Fluorescent molecular imaging can help identify such lesions, but conventional imaging probes produce high background signal which can obscure peritoneal implants. To achieve high sensitivity and specificity, a two-step activatable fluorescent probe set was synthesized in which the tumor is first pretargeted with a biotinylated antibody and then a subsequently administered optical probe, neutravidin-BODIPY-FL, is activated via the molecular interaction of the fluorophore with biotin. A single antibody can be loaded with more than 10 biotin molecules. The fluorescence of the probe is increased approximately 10-fold after it binds to the biotinylated antibody leading to high sensitivity for peritoneal implants and reduced background signal of the unbound probe. Using this two-step pretargeting strategy, it was possible to detect submillimeter nodules disseminated within the peritoneal cavity in a mouse model of ovarian cancer with high sensitivity and specificity. This two-step activation strategy for molecular optical imaging is potentially widely applicable to other tumor types and other anatomic sites. For details, see the article by Hama et al. on page 3809 of this issue.