Precise and reproducible isolation of desired cell types or layers from heterogeneous tissues is crucial to analyze specific gene profiles and molecular interactions in vivo. Forebrain is the core site of higher functions, like cognition and memory consolidation. It is composed of heterogeneous and distinct cell types, interconnected to form functional neural circuits. Any alteration in the development or function often leads to brain disorders with profound consequences. Thus, precise molecular understanding of forebrain development in normal and diseased scenarios is important. For quantitative studies, most traditional analytical methods require pooling of large cell populations, that results in loss of in vivo tissue integrity and of spatial, molecular and cellular resolution. Laser capture microdissection (LCM) is a fast and extremely precise method of obtaining uncontaminated, homogeneous sets of specific cell types and layers. Our current procedure involves cryo-sectioning and laser microdissection of fresh-frozen mouse forebrains, that are genetically modified and treated with small-molecule therapeutics. Using LCM, specific regions of interest, such as neural layers, cells from adjacent yet distinct subregions within a tissue layer, are obtained under RNase-free conditions. These small cellular cohorts are further used for downstream, high-throughput genomic or transcriptomic assays. Here, we have introduced break-points at multiple stages throughout our protocol. This makes our method simpler and more user-friendly to follow, without compromising on the quality. The current protocol can easily be adapted for different brain regions, as well as for other model organisms/human tissue.