Gold nanoparticles (AuNPs) are promising imaging agents for the long-term visualization and tracing of intracellular functions because they bear outstanding optical properties and are fairly easily bioconjugated. However, the design of such multifunctional nanosystems might be limited by their bioavailability. Cell-penetrating peptides (CPPs) have been shown to be efficient molecular transporters with very few indices of cytotoxicity also in conjunction with nanoparticles. In this context, the current work aims to explore the approach of in situ conjugation during laser ablation in liquids for the design of CPP−NP conjugates at the example of penetratin-conjugated AuNPs. Because penetratin is positively charged at neutral pH, the conjugation process most likely differs from the previously reported coupling of oligonucleotides with their negatively charged phosphate backbone. Results reveal that penetratin is more efficiently bound to AuNPs, increasing the pH value of the ablation media, whereas the size and morphology of the bioconjugates function in terms of the penetratin concentration during the laser process. Probable underlying processes such as size quenching, aggregation, and laser-induced partial melting are assessed by the means of transmission electron microscopy and UV−vis spectroscopy. In a preliminary biological study, laser scanning confocal and transmission electron microscopy revealed a successful uptake of penetratin-conjugated AuNPs for the first time in up to 100\% of coincubated cells within 2 h.