This review article discusses the mechanisms of cellular uptake of cell-penetrating peptides (CPPs), which are short, cationic peptides capable of transporting large cargo molecules into cells. CPPs can enter cells through two main mechanisms: direct penetration and endocytosis. Direct penetration involves membrane translocation without energy consumption, while endocytosis includes endocytic entry followed by endosomal escape. The exact molecular mechanisms remain debated, with factors such as CPP concentration, sequence, and lipid composition influencing the uptake process. CPPs are categorized into three main types: primary amphipathic (paCPPs), secondary amphipathic (saCPPs), and nonamphipathic (naCPPs). paCPPs, such as transportan and TP10, are more hydrophobic and can penetrate membranes directly. saCPPs, like penetratin and pVEC, are amphipathic and can interact with lipid membranes. naCPPs, such as R9 and TAT, are less toxic and rely more on endocytosis. Various methods, including fluorescence imaging, confocal microscopy, and functional assays, have been used to study CPP uptake mechanisms. These methods help determine whether CPPs enter cells via direct penetration or endocytosis. Inhibitors and model systems have also been employed to investigate the role of different pathways. Factors affecting CPP uptake include the CPP's physicochemical properties, such as hydrophobicity, charge, and sequence, as well as experimental conditions like temperature and cell type. The presence of cargo can also influence the uptake mechanism, with some cargos requiring endocytosis for efficient delivery. Selected CPPs, such as TAT, penetratin, and pVEC, have been studied in detail. TAT is known for its ability to deliver cargo into cells, while penetratin and pVEC are involved in endocytic pathways. TP10, a hydrophobic CPP, can cause membrane leakage at low concentrations. CPPs and antimicrobial peptides (AMPs) share structural similarities and both can interact with cell membranes. However, CPPs are generally less toxic and can deliver cargo without significant membrane damage. The mechanisms of action for both CPPs and AMPs are not fully understood, but they may involve pore formation, membrane perturbation, or endocytosis. In conclusion, CPPs are valuable tools for delivering cargo into cells, with mechanisms varying depending on the CPP type and experimental conditions. Further research is needed to fully understand the molecular mechanisms and optimize their use in drug delivery and therapeutic applications.This review article discusses the mechanisms of cellular uptake of cell-penetrating peptides (CPPs), which are short, cationic peptides capable of transporting large cargo molecules into cells. CPPs can enter cells through two main mechanisms: direct penetration and endocytosis. Direct penetration involves membrane translocation without energy consumption, while endocytosis includes endocytic entry followed by endosomal escape. The exact molecular mechanisms remain debated, with factors such as CPP concentration, sequence, and lipid composition influencing the uptake process. CPPs are categorized into three main types: primary amphipathic (paCPPs), secondary amphipathic (saCPPs), and nonamphipathic (naCPPs). paCPPs, such as transportan and TP10, are more hydrophobic and can penetrate membranes directly. saCPPs, like penetratin and pVEC, are amphipathic and can interact with lipid membranes. naCPPs, such as R9 and TAT, are less toxic and rely more on endocytosis. Various methods, including fluorescence imaging, confocal microscopy, and functional assays, have been used to study CPP uptake mechanisms. These methods help determine whether CPPs enter cells via direct penetration or endocytosis. Inhibitors and model systems have also been employed to investigate the role of different pathways. Factors affecting CPP uptake include the CPP's physicochemical properties, such as hydrophobicity, charge, and sequence, as well as experimental conditions like temperature and cell type. The presence of cargo can also influence the uptake mechanism, with some cargos requiring endocytosis for efficient delivery. Selected CPPs, such as TAT, penetratin, and pVEC, have been studied in detail. TAT is known for its ability to deliver cargo into cells, while penetratin and pVEC are involved in endocytic pathways. TP10, a hydrophobic CPP, can cause membrane leakage at low concentrations. CPPs and antimicrobial peptides (AMPs) share structural similarities and both can interact with cell membranes. However, CPPs are generally less toxic and can deliver cargo without significant membrane damage. The mechanisms of action for both CPPs and AMPs are not fully understood, but they may involve pore formation, membrane perturbation, or endocytosis. In conclusion, CPPs are valuable tools for delivering cargo into cells, with mechanisms varying depending on the CPP type and experimental conditions. Further research is needed to fully understand the molecular mechanisms and optimize their use in drug delivery and therapeutic applications.