In order to visualize the effects of aqueous surfactant-humectant systems on the skin barrier, an in vitro two-photon fluorescence microscopy (TPM) study, including dual-channel visualization, was carried out. TPM is a non-invasive imaging technique based on two-photon induced nonlinear excitations of fluorophores, with the capability for deep-tissue imaging (up to several hundred micrometers). The following aqueous solutions of surfactants, a humectant, and a surfactant+humectant mixture that contacted pig full-thickness skin (p-FTS) were studied: (i) a harsh surfactant solution-sodium dodecyl sulfate (SDS) (1 wt%); (ii) a harsh surfactant+humectant solution-SDS (1 wt%) + glycerol (10 wt%); (iii) a mild surfactant solution-sodium cocoyl isethionate (SCI) (1 wt%); (iv) a control solution-phosphate-buffered saline (PBS); and (v) a humectant solution-glycerol (10 wt%). Sulforhodamine B (SRB), a hydrophilic fluorescent probe, was used to visualize the effects of aqueous contacting solutions i-v on the skin barrier morphology. The results of the TPM visualization study revealed that SDS induces corneocyte damage by denaturing keratins and creating intracorneocyte penetration pathways. On the other hand, SDS+glycerol did not significantly induce corneocyte damage. The dual-channel TPM images corresponding to aqueous contacting solutions iii-v showed low SRB penetration into the corneocytes, as well as localization of the SRB probe within the lipid bilayers surrounding the corneocytes of the SC. Through a quantification of the amount of SRB that penetrated into the skin as a function of skin depth, we found that adding glycerol to an SDS aqueous contacting solution can significantly reduce the SDS-induced penetration depth of SRB, which provides evidence of the ability of glycerol to mitigate SDS-induced skin barrier perturbation. The distribution of SRB in the p-FTS samples was analyzed using a theoretical model that quantified changes in the skin aqueous pore characteristics induced by aqueous contacting solutions i, ii, iii, and v, relative to aqueous contacting solution iv, the control. The results of the theoretical model indicate the following ranking order in the extent of perturbation to the skin aqueous pores (from the highest to the lowest): i > ii > iii > iv > v. The development of such an in vitro visual ranking methodology, including quantification using TPM, can potentially reduce many costly in vivo screening procedures, thereby significantly reducing the cost and time-to-market of new cosmetic formulations containing surfactants and humectants.