Kahalalide F (KF) is a natural product currently under phase II clinical trials. Here, we report the solid phase synthesis of 132 novel analogues of kahalalide F and their in vitro activity on a panel of up to 14 cancer cell lines. The structure-activity relationship of these analogues revealed that KF is highly sensitive to backbone stereotopical modification but not to side chain size modification. These observations suggest that this compound has a defined conformational structure and also that it interacts with chiral compounds through its backbone and not through its side chains. The N-terminal aliphatic acid appears to be a hydrophobic buoy in a membrane-like environment. Moreover, significant improvement of the in vitro activity was achieved.