Cell-penetrating peptides have been widely used as a tool to gain access to cytosol for numerous applications. The review highlights the advances made in preclinical and clinical research using cell-penetrating peptides since their discovery in 1980s. Further, the emphasis is on summarizing the role of cell-penetrating peptides as permeation enhancers for transdermal and topical drug delivery applications. A summary table of preclinical studies utilizing various peptides in combination with different active ingredients and drug delivery systems is included. Lastly, we capture the challenges associated with the cell-penetrating peptides to translate the preclinical work to clinical applications.