Introduction: Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non-adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Furthermore, this systems have the additional advantage of using low amounts of reagents and cells. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell-derived sphere is an attractive alternative to identify CSCs. The objective of this study, was to test the effectiveness of microdevices, analyzing the formation of tumorspheres from single cell division, and to evaluate the heterogeneity within CSC populations and its interaction with different components of the extracellular matrix, using MB49-I bladder cancer cell line. The results show the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under low adhesion conditions. Plating increasing number of cells, from 2 cell/ ul to 64 cell /ul, we determined 32 cells / ul, as the optimal initial number, which allowed the development of spheres at 14 days. We observed that the volume of reagents used in the microdevice resulted in 29 times lower compared to traditional plates of cell culture. In addition, the device allowed the tracking of single cells derived spheres, observing different patterns of growth, resulting in large, medium, and small spheres, indicating heterogeneity within the cell line and different stages in the cell population. We also determined by immunofluorescence the expression of Oct4 and CD44, known CSC markers. Furthermore, the division of CSCs in different matrices was studied, as a preliminary analysis for the differential selection of tumorspheres in future studies. We observed that spheres were able to grow in collagen IV. On the contrary, in matrigel cell differentiation was observed. In conclusion, we present a LOC device that allowed the study and identification of CSC of bladder tumor cells with the advantage of using minimum amount of reagents and samples. The present device promises to be considered as a new useful technology to be used as a complement or replacement of traditional studies in the culture of tumor cells.