Gene transfection continues to be a major challenge in chemistry, biology and materials sciences. New methodologies and recent breakthroughs have renewed the interest in the discovery and development of new tools for efficient gene transfection. Hydrazone formation between a cationic head and hydrophobic tails has emerged as one of the most promising techniques for nucleotide delivery. In this contribution, we have exploited hydrazone formation to modulate the transfection activity of a parent linear peptide in combination with a plasmid DNA cargo. This strategy allowed the straightforward preparation, under physiologically compatible conditions, of a discrete library of amphiphilic modulated penetrating peptides. Without the requirement of any isolation or purification steps, these modulated amphiphilic peptides were combined with a plasmid DNA and screened in transfection experiments of human HeLa cells. Three of these hydrazone-conjugated peptides were identified as excellent vectors for plasmid delivery with comparable, or even higher, efficiencies and lower toxicity than the commercial reagents employed in routine transfection assays.