The cytotoxic activity of (-)-chlorizidine A, a marine alkaloid containing a unique fusion between a pyrroloisoindolone and dehydropyrrolizine, was explored using a combination of cellular and molecular methods. Our studies began by applying preliminary SAR evidence gathered from semisynthetic bioactivity evaluations, to prepare an active immunoaffinity fluorescent (IAF) probe. This probe was then used to identify two cytosolic proteins, GAPDH and hENO1, as the targets of (-)-chlorizidine A.