A new type of monoclonal antibody (mAb)-based, highly specific phototherapy (photoimmunotherapy; PIT) that uses a near infrared (NIR) phthalocyanine dye, IRDye700DX (IR700) conjugated with a mAb, has recently been described. NIR light exposure leads to immediate, target-selective necrotic cell death in vitro. Detecting immediate in vivo cell death is more difficult because it takes at least 3 days for the tumor to begin to shrink in size. In this study, fluorescence lifetime (FLT) was evaluated before and after PIT for monitoring the immediate cytotoxic effects of NIR mediated mAb-IR700 PIT. Anti-epidermal growth factor receptor (EGFR) panitumumab-IR700 was used for targeting EGFR-expressing A431 tumor cells. PIT with various doses of NIR light was conducted in cell pellets in vitro and in subcutaneously xenografted tumors in mice in vivo. FLT measurements were obtained before and 0, 6, 24, and 48 hours after PIT. In vitro, PIT at higher doses of NIR light immediately led to FLT shortening in A431 cells. In vivo PIT induced immediate shortening of FLT in treated tumors after a threshold NIR dose of 30 J/cm(2) or greater. In contrast, lower levels of NIR light (10 J/cm(2) or smaller) did not induce shortening of FLT. Prolongation of FLT in tissue surrounding the tumor site was noted 6 hours after PIT, likely reflecting phagocytosis by macrophages. In conclusion, FLT imaging can be used to monitor the acute cytotoxic effects of mAb-IR700-induced PIT even before morphological changes can be seen in the targeted tumors.