This study aims to utilize three-dimensional (3D) Raman imaging technology for the stratified quantification of active ingredients (AIs) within the skin, which involves determining their contents in the stratum corneum (SC) and the active epidermal (EP) layer. Multiple linear regression combined with a least squares algorithm was used to analyze the relative concentration coefficients of targets in the skin. However, an issue of signal attenuation with increasing depth was encountered during the 3D Raman data acquisition process. A calibration process was performed based on establishing the relationships among depth in skin, the relative concentration coefficients of a two-dimensional (2D) Raman imaging and the concentration value from high-performance liquid chromatography (HPLC) technology. Nicotinamide was selected as the target in this study, and a 3D Raman signal attenuation correction equation specific to nicotinamide was developed. A 3D stratified imaging of nicotinamide in skin was achieved by this method. This method offers a visual analysis of AIs in the SC and EP by in vivo Raman technology.