Common workflows in bottom-up proteomics require homogenization of tissue samples giving to get access to the biomole-cules within the cells. The homogenized tissue samples often contain many different cell types, thereby representing an average of the natural proteome composition and rare cell types are not sufficiently represented. To overcome this problem, small vol-ume sampling and spatial resolution is needed to maintain a better representation of the sample composition and their proteo-me signatures. Using nanosecond infrared laser ablation, the region of interest can be targeted in a three dimensional (3D) fash-ion whereby the spatial information is maintained during the simultaneous process of sampling and homogenization. In this study, we ablated 40 µm thick consecutive layers directly from the scalp through the cortex of the embryonic mouse heads and analyzed them by subsequent bottom-up proteomics. Extra- and intracranial ablated layers showed distinct proteome profiles comprising expected cell specific proteins. Additionally, known cortex markers like SOX2, KI67, NESTIN and MAP2 showed a layer specific spatial protein abundance distribution. We propose potential new marker proteins for cortex layers such as MTA1 and NMRAL1. The obtained data confirm that the new 3D -tissue sampling and homogenization method is well suited for investigating the spatial proteome signature of tissue samples in a layer-wise manner. Characterization of the proteome composition of embryonic skin and bone structures, meninges, and cortex lamination in situ, enables to a better understanding of molecular mechanisms of development during embryogenesis and disease pathogenesis.