In the last three decades, many new cell‐penetrating peptides (CPPs) were developed that exhibited enhanced cell selectivity. Thus, we aimed to validate the tumor cell selectivity of peptides from this new generation, namely fragments mini‐crotamine and mini‐maurocalcine. Both of these peptides are derived from venoms. Furthermore, we studied an analog of the classical CPP HIV‐TAT(47‐57) with alternating chirality of Arg residues. To allow covalent coupling of cargoes or fluorophores, a cysteine residue was introduced to the N‐terminus of the synthesized peptides. The therapeutic antibody trastuzumab conjugated to different fluorescent dyes was used for internalization studies. Comparison of uptake efficiencies revealed that CPPs of the new generation are in contrast to MPG‐peptides, nearly unable to internalize the noncovalently formed complexes with trastuzumab. Interestingly, the fluorescent derivative of the crotamine fragment was mainly observed in a subpopulation of breast cancer cells, whereas it was homogenously distributed in fibrosarcoma, colon cancer, and noncancerous endothelia cells. Thus, the fluorescent crotamine fragment reported herein is a potent theranostic tool for image‐guided applications. This peptide can be used to pinpoint the level of heterogeneity present within tumors and aid in the generation of therapeutics that target heterogenic subpopulations.