The advent of spatially resolved transcriptomics has revolutionized our understanding of tissue heterogeneity. While advanced spatial transcriptomics (ST) technologies offer unparalleled and unbiased, whole-slide molecular mapping, laser microdissection (LMD) remains a highly valuable and often superior approach for specific research questions, particularly when coupled with RNA sequencing (RNA-seq). LMD provides high spatial resolution through precise morphological annotation, allowing for the isolation of defined cell clusters or even rare populations from complex tissues. This enables deeper and more sensitive transcriptomic profiling than is often achievable with some high-throughput ST methods that depend on computational deconvolution. Crucially, LMD is well established for use with routine histological samples, including challenging formalin-fixed paraffin-embedded (FFPE) specimens, facilitating invaluable retrospective studies on large patient cohorts. Unlike emerging ST platforms that can present significant per-sample costs and substantial computational overhead, targeted LMD-RNA-seq offers a more cost-effective and scalable solution for analyzing numerous specific regions across large numbers of patients. Therefore, a robust protocol outlining the use of LMD for routine histological samples, coupled with RNA-seq, remains an invaluable tool for discovery, enabling highly specific molecular insights from clinically relevant biospecimens.