Crossing through the stratum corneum is the rate limiting step in the permeation of skincare active ingredients into the underlying epidermis and beyond. While numerous approaches can be employed to enhance the permeation of active ingredients, tracking and profiling the permeation of these ingredients through the stratum corneum can be challenging, particularly if the chemistry of the active ingredient is similar to that of the native chemistry of the skin. Here, the state-of-the-art 3D OrbiSIMS technique has been used to demonstrate the permeation of the biologically active anti-ageing peptide Pal-GHK into the stratum corneum. 3D OrbiSIMS combines secondary ion mass spectrometry with the high mass-resolving power of an Orbitrap^TM mass analyser, facilitating in situ label free molecular analysis and the identification of organic species in complex solid samples, including biological tissues [Passarelli et al., Nat. Methods; 2017; 14: 1175-1183]. A 3D OrbiSIMS approach was used to determine the Pal-GHK peptide content in vivo and ex vivo through tape strips collected from the human volar forearm stratum corneum and using full thickness human skin respectively. For the in vivo study, twelve subjects were treated with formulations containing <100 ppm Pal-GHK applied at 2mg/cm² to the volar forearms, with one area remaining untreated. Four hours post-application, 15 sequential tape strips were collected from all sites using D-squame^® tape strips. The optical absorption of the tape strips was measured in triplicate at 850 nm to determine skin cell density. 3D OrbiSIMS was then used to determine the presence and intensity of the Pal-GHK peptide at 3 separate points on the tape strip. For ex vivo analysis, human skin (removed during cosmetic surgery) was mounted, dermal side down, in a Franz-type static diffusion cell set-up. Infinite doses of formulations containing Pal-GHK at <100 ppm were applied to the donor chamber, with the skin exposed to the formulations for 4 hours. After 4 hours, the Franz cell was dismantled, excess formulation was removed with a dry sponge and the skin was dehydrated under vacuum at room temperature for 24 hours before 3D OrbiSIMS analysis. Throughout the analysis, the Pal-GHK molecular ion, C₃₀H₅₅N₆O₅⁺, was used as the diagnostic marker, having been confirmed to be present and absent in the formulations and untreated tape strips respectively. The depth profile of Pal-GHK across 15 stratum corneum tape strips was then determined, and further analysis of the data showed that Pal-GHK could be clearly detected in the 10^th tape strip and beyond, demonstrating significant penetration (p<0.05, versus untreated control) to at least 10 layers into the skin surface. In addition, ex vivo Franz-cell experiments demonstrated that the Pal-GHK molecular ion was clearly detected, with the ion intensity decreasing with increasing skin depth, reaching a baseline level at the stratum corneum – epidermal junction. Despite the low concentration of Pal-GHK within the skin and its chemical similarity to native skin components, this novel approach clearly identified the molecular ion and facilitated assessment of the permeation of the peptide as a function of skin depth; data that has previously been difficult to accurately attain. 3D OrbiSIMS has enormous potential to provide molecular information for skin research and further understanding of active ingredient delivery.