While the cellular cytosol and organelles contain attractive targets for disease treatments, it remains a challenge to deliver therapeutic biomacromolecules to these sites. This is due to the selective permeability of the plasma and endosomal membranes, especially for large and hydrophilic therapeutic cargos such as proteins and nucleic acids. In response, many different delivery systems and molecules have been devised to help therapeutics cross these barriers to reach cytosolic targets. Among them are peptide and protein-based systems, which have several advantages over other natural and synthetic materials including their ability to interact with cell membranes. In this review, we will describe recent advances and current challenges of peptide and protein strategies that leverage cell membrane association and modulation to enable cytosolic delivery of biomacromolecule cargo. The approaches covered here include peptides and proteins derived from or inspired by natural sequences as well as those designed for delivery function.