This paper presents a tubing-free microfluidic wound healing assay to quantify the migration of vascular smooth muscle cells (VSMCs), where gravity was used to generate a laminar flow within microfluidic channels, enabling cell seeding, culture, and wound generation. As the first systemic study to quantify the migration of VSMCs within microfluidic environments, the effects of channel geometries, surface modifications and chemokines on cellular migration were investigated, revealing that 1) height of the micro channels had a significant impact on cell migration; 2) the surface coating of collagen induced more migration of VSMCs than fibronectin coated surfaces and 3) platelet derived growth factor resulted in maximal cell migration compared to tumor necrosis factor alpha and fetal bovine serum. Furthermore, migrations of five types of VSMCs (e.g., the human vascular smooth muscle cell line, two types of primary vascular smooth cells, and VSMCs isolated from two human samples) were quantified, finding that VSMCs from the cell line and human samples demonstrated comparable migration distances, which were significantly lower than the migration distances of two primary cell types. As a platform technology, this wound healing assay may function as a new model to study migration of VSMCs within microfluidic environments.