Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well‐defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in‐depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one‐dimensional SDS‐PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself.