Digital spatial profiling (DSP) is a nondestructive method for high-plex spatial profiling of proteins and RNA from a wide variety of sample types, including formalin-fixed, paraffin-embedded (FFPE) tissue sections. This method uses small photocleavable oligonucleotide “barcodes” (PC-oligos) covalently attached to in-situ affinity reagents (antibodies and RNA-probes) to provide unlimited multiplexing capability. The photocleavage light is projected onto the tissue slice using two-digital micromirror devices (DMD), containing one-million semiconductor-based micromirrors allowing complete flexibility in the pattern of light utilized for high-plex digital profiling of the tissue. These spatial light-patterns can be automatically configured to profile (1) “tumor-only” cells plus “tumor-microenvironment-only” cells; (2) unique cell types and rare cell features (e.g., macrophages, CD8, CD3, CD45, PD-L1 on macrophages, PD-L1 on tumors, etc.); (3) spatial gradients around cell-features or tumor features (e.g., excluded boundaries); (4) hypothesis-free spatial grids; (5) simple hand-selected geometric areas (e.g., free-hand software-based “drawing” on tissue regions); and (6) or any combination of the above modalities. These DMDs can automatically configure themselves to “align” to the biology presented by each individual tissue section. Advanced validated high-plex panels of proteins (~100-plex) and RNA (up to 20,000-plex) specifically designed for immuno-oncology (IO) have been developed. Immuno-oncology clinical trial samples examined using DSP have already provided key insights into the mechanism of action of combination therapy in melanoma, appearing recently in back-to-back articles published in Nature Medicine. DSP has been developed with knowledge of key immuno-oncology terms (tumor, tumor microenvironment, stroma, etc.) and prevalidated high-plex panels of affinity markers (antibodies and in situ RNA probes) and has the potential to bring the full power of high-plex molecular profiling to spatially resolved studies.