Conventional transcriptomics analyzes a mixture of cells simultaneously either from tissue or single cells, for which the information on its in vivo or in situ position is lost. Although we can now perform cell type annotations for heterogenous samples using single-cell profiling, information on the spatially defined attributes that underpin the heterogeneous nature of the cells is lacking. Here, we describe a further optimized 10× Visium spatial transcriptomics protocol, a method that enables the deconvolution of heterogenous tissues while retaining its spatial information. The 10× Visium protocol allows the transcriptomic profiling of both fresh frozen and FFPE tissues while retaining their native spatial information. This protocol has wide potential applications to address the intricacies of the tumor microenvironment such as cellular interactions between the different tissue compartments and or with immune cells and elucidate how those interactions may contribute to tumorigenesis or disease progression. The 10× Visium spatial transcriptomics assay has been widely applied to study the spatial transcriptome of various tissue types across human and mouse samples such as lung, melanoma, and endometrial cancer to name a few. The entire protocol from tissue collection to final library preparation for fresh frozen samples takes about 5 days and for FFPE samples, about 3 days. Sequencing time ranges from 2 to 4 weeks and varies between different sequencing vendors.