Protein N-termini represent a wealth of biological information, such as their generation by limited proteolysis, which sculpts proteome functionality by yielding protein-sized cleavage products with novel functionality. N-terminomics is a branch of functional proteomics and enables the analysis of thousands of protein N-termini in an unbiased manner. By targeting native, proteolytically generated or acetylated protein N-termini as well as uncovering novel translation initiation sites, N-terminomics bears relevance to proteolysis and proteogenomics research. Unlike proteomic assays to study modifications such as phosphorylation, N-terminomics has remained inaccessible to non-expert laboratories due to a lack of ready-to-use kit formats or commercial services. Based on a recently published N-terminomic workflow by my laboratory, the present project aims to fill this niche by laying the foundation to represent N-terminomics (sample preparation up to mass spectrometry analysis) in a kit-like format together with online data analysis. Development steps include inclusion of multiple internal standards for improved sample-to-sample comparability, sole usage of cost-effective, disposable “small-scale” equipment in sample preparation, minimization of required input material, adaption to different input material including cryopreserved and formalin-fixed, paraffin-embedded samples, as well as web-based mass spectrometry data analysis and annotation. This endeavor is based on multiple collaborative projects of the applicant with pharmaceutical companies and backed by interest of proteomic supply companies. In a small forward-looking activity, the project will explore the application N-terminomics in molecular diagnostics of bladder cancer; based on the rationale local infiltration by solid tumors is based on differential proteolysis. This activity serves an initial step to add N-terminomics to the toolbox of molecular pathology.