Hexetidine is utilized in antimicrobial preservative products that are rinsed off at a maximum concentration of 0.1% in both Korea and the European Union (EU). As no reports exist regarding the dermal absorption of hexetidine in cosmetic products, an in vitro dermal absorption study was conducted in compliance with the guideline according to the Ministry of Food and Drug Safety (MFDS) of Korea. Prior to conducting the dermal absorption study, analytical methods were established using liquid chromatography-mass spectrometry (LC-MS/MS) with a multiple reaction monitoring (MRM) technique for the quantification of hexetidine in various matrices, including swabs, stratum corneum (SC), skin (dermis and epidermis), and receptor fluid (RF). The hexetidine analytical method used in this study demonstrated excellent linearity (r² = 0.9944-0.9999), accuracy (80.9-108.3%), and precision (0.6-16.1%) in compliance with the validation guidelines. To evaluate the skin absorption of hexetidine in hair products, a Franz diffusion cell was utilized. 0.1, 0.025% gel shampoo formulation (wash-off) of hexetidine was applied to mini-pig skin at a dose of 10 μl/cm². Following a 30-minute application period, an interim wash-out step was performed to simulate shampoo conditions for the gel formulation. After 24 hours of application, the skin was wiped with a swab, and the stratum corneum (SC) was collected using tape stripping. Subsequently, the mini-pig skin was sectioned into eight pieces, and receptor fluid (RF) samples were collected at 0, 1, 2, 4, 8, 12, and 24 hours. As a result, the total dermal absorption rate of hexetidine was determined to be 0.01 ± 0.01 μg/cm² (0.07 ± 0.06%) and 0.00 ± 0.00 μg/cm² (0.00± 0.00%) for the gel shampoo formulations, respectively. These results can be utilized for the exposure assessment of hexetidine when used as a cosmetic ingredient.