In the search of biomaterials that promote cell adhesion, it is crucial to explore the integrin-substrate dynamic interactions given in a certain cell type to design successful biofunctionalization strategies. Here, we use a microarray platform for a thorough characterization of cell adhesion to a particular substrate. A biosensor based on an array of 20 μm fibronectin circular isles was adapted to tissue culture treated plates to facilitate the performance of cell adhesion assays and the posterior affinity analyses. This sensitive analytical tool enables not only the evaluation of the cell adhesion kinetics, but also the integrin profiling and their contribution to cell attachment and adhesion strengthening via clustering. In particular, the biosensor was able to detect a significantly slower adhesion kinetics in fibroblasts, namely Baby Hamster Kidney Fibroblasts (BHK) and Human Dermal Fibroblasts (hDF), in comparison to other cell types such as C2C12 Mouse Myoblasts (C2C12) or Human Mesenchymal Stem Cells (hMSCa). When directly comparing hDF and hMSCa, the analysis determined that the differing kinetics were caused by a distinct integrin expression profile. Whereas β1-presenting integrins were the major responsible for hDF attachment, hMSCa adherence was importantly dependent on β1 but also on other integrin classes. Additionally, results revealed that concerning cell adhesion consolidation, in hMSCa, both αvβ3 and β1-subunit-presenting integrins contributed similarly; whereas in hDF, the latter played a more important role. Hence, our biosensor provided crucial information for the development of new cell-adhesive biomaterials, which are key in multiple biomedical fields including cell therapy or tissue engineering.