The synthetic peptide TAT-I24 (GRKKRRQRRRPPQCLAFYACFC) exerts antiviral activity against several double-stranded (ds) DNA viruses, including herpes simplex viruses, cytomegalovirus, some adenoviruses, vaccinia virus and SV40 polyomavirus. In the present study, in vitro profiling of this peptide was performed with the aim of characterizing and improving its properties for further development. As TAT-I24 contains three free cysteine residues, a potential disadvantageous feature, peptide variants with replacements or deletions of specific residues were generated and tested in various cell systems and by biochemical analyses. Some cysteine replacements had no impact on the antiviral activity, such as the deletion of cysteine 14, which also showed improved biochemical properties, while the cyclization of cysteines 14 and 20 had the most detrimental effect on antiviral activity. At concentrations below 20 µM, TAT-I24 and selected variants did not induce hemolysis in red blood cells (RBCs) nor modulated lipopolysaccharide (LPS)-induced release of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in human peripheral blood mononuclear cells (PBMCs). These data indicate that TAT-I24 or its peptide variants are not expected to cause unwanted effects on blood cells.