We report in this work the discovery of novel allosteric MEK inhibitors by pharmacophore modeling and virtual screening. Two out of 13 virtual hit compounds were identified as MEK kinase inhibitors using a MEK1 binding assay. Structural derivations on the hit compound M100 (IC50 = 27.2 ± 4.5 μM in RAF-MEK cascading assay) by substituent transformation and bioisosterism replacement have led to the synthesis of a small library of carbazoles. The enzymatic studies revealed the preliminary structure-activity relationships and the derivative 22k (IC50 = 12.8 ± 0.5 μM) showed the most potent inhibitory effect against Raf-MEK cascading. Compound 7 was discovered as toxic as M100 to tumor cells whereas safer to HEK293 cells (IC50 > 100 μM) than M100 (IC50 = 8.9 ± 2.0 μM). It suggests that carbazole is a good scaffold for the design of novel MEK inhibitors for therapeutic uses. More importantly, the developed pharmacophore model can serve as a reliable criterion in novel MEK inhibitor discovery.