In this study, a series of tetrahydroisoquinolines have been synthesized and identified as novel kinesine spindle protein (KSP) inhibitors based on the pharmacophore we have mapped and the crystal structure of monastrol bound to the target protein. The KSP inhibitory activities of all the designed compounds were tested using cloned Human KSP protein. All thirteen compounds were more potent than the control, monastrol, in Human KSP protein adenosine triphosphatase (ATPase) assays. Three compounds (1b, 1g, 1h) exhibited over 100 times higher potency than monastrol. Cytotoxic results in vitro by MTT method indicated that nine of these compounds (1a, 1b, 1c, 1d, 1e, 1g, 1h, 1j, 1k) were more active than monastrol. In particular, compounds 1b and 1g, each of which contains a hydrophilic group on the side chain at the 2-position, exhibited excellent cell-killing activities against HepG2 cells.