For many classes of peptide, protein and nucleic acid drugs, intracellular delivery remains the primary obstacle in drug development. This obstacle has been challenging because many methods that are used to measure “cell penetration” actually measure total cellular uptake, including material trapped at the cell surface or in endosomes. For a long time, methods for measuring cytosolic penetration were not accessible, and this has greatly slowed the development of several classes of biotherapeutics. Further, without basic understanding of cytosolic penetration in a time-resolved, cell-type-specific manner, we will never move beyond trial- and-error as a means for developing biotherapeutics for specific disease targets in specific tissues. In previous work, we developed the ChloroAlkane Penetration Assay (CAPA) to solve some of the problems of previous methods. CAPA has been adopted by a large number of academic and industrial labs, and it is becoming a new “gold standard” for measuring cytosolic penetration. In this renewal proposal, we describe new opportunities to address challenging problems in biotherapeutics development. The first is how to measure cytosolic penetration in different cell types. In Aim 1, we describe using adeno-associated viruses (AAVs) to enable the versatile CAPA assay in any cell type of interest. This unlocks exciting new opportunities to compare cytosolic penetration across dozens of cell lines, including primary cells, and to allow measurement of penetration to different subcellular compartments. A second important problem is to understand the kinetics of cytosolic penetration. In Aim 2, we solve this problem by introducing a new “turn-on” version of CAPA, which should be more sensitive and allow real-time measurements. Finally, in Aim 3, we envision a ChloroAlkane Penetration Screen (CAPS) which can screen pooled libraries of thousands to millions of molecules at a time. We will apply this new screen to large libraries of cyclic peptides and antisense oligonucleotides. These data will represent a huge leap in our understanding of structure- penetration relationships for these classes of molecules, and the new screen will be incorporated into a design- test-learn cycle to produce data-driven design algorithms for cytosol-penetrant molecules. This project is well-suited for PAR-19-253, Focused Technology Research and Development, because it is focused on innovative methods. These new methods will allow for measuring a molecule’s penetration in any cell, to any compartment, and in real-time. They will also allow for custom screens of millions of molecules to optimize cytosolic penetration, and they will provide data-driven rules for designing better oligonucleotide and peptide drugs. Like CAPA, these new methods are designed to be simple and accessible, so that they can be widely adopted by researchers working on peptide, protein, and nucleic acid therapeutics.