Success in tissue engineering requires an understanding of how cells integrate the signals presented from the microenvironment created by biomaterial scaffolds to alter their responses. Besides the presence of chemical stimuli, there is growing evidence that the spatial organization of cells and tissue within a 3-dimensional (3-D) extracellular matrix (ECM) context is a critical element in controlling cellular function. Therefore, in order to direct cells toward a desirable tissue structure, it is necessary to engineer biomaterials to have spatiotemporal control of the presentation of regulatory signals. Given that, micro-patterning techniques have profited by combining micro-fabrication technology with the chemical conjugation of biologically active molecules to provide new culture systems where cells can be cultured within a specific geometry. The micro-engineered environments have been developed as 2- and 3-D structures, which have proven greatly useful as versatile platforms to study cell, biomaterial, and ECM interactions on both macroscopic and microscopic levels. The main focus of this review is a brief summary of the use of micro-engineered substrates in the analysis of cell-biomaterial interactions with the aim to provide an introductory overview of practical applications available in the literature. In particular, topics regarding (1) the soft-lithography technique to prepare micro-patterned substrates for the spatial control of cell adhesion, (2) biomaterials stiffness-dependent cellular responses, and (3) the microarray techniques for analysis of cell/biomaterials interactions are discussed.