The TLC profiles of intra- and inter-day precision for Piper betle L. (PBL) folium methanol extract was studied for their peak marker recognition and identification. The Numerical chromatographic parameters (NCPs) of the peak markers, the hierarchical clustering analysis (HCA) and the principal component analysis (PCA) were applied to authenticate the PBL. folium extract from other Piper species folium extract and to ensure the antifungal activity quality of the PBL essential oil. The spotted extract was developed with the mobile phase of toluene: ethyl acetate; 93:7, (v/v). The eluted plate was viewed with the TLC-Visualizer, scanned under absorption and fluorescent mode detection, and on each sample the in-situ UV spectra were recorded between 190 to 400 nm. The NCPs profiles of intra- and inter-day precision results offered multi-dimensional chromatogram fingerprints for better marker peak pattern recognition and identification. Using the r-value fingerprints data series generated with this method allowed more precise discrimination the PBL. from other Piper species compared to the marker peak area fingerprint method. The cosine pair comparison was a simple method for authentication of two different fingerprints. The ward linkage clustering and the pair cross-correlation comparison were better chemometric methods to determine the consistency peak area ratio between fingerprints. The first component PCA-loading values of peak marker area fingerprints were correlated linearly to both the bio-marker concentration as well as the antifungal activity. This relationship could be used to control the quality and pharmacological potency. This simple method was developed for the authentication and quantification of herbal medicine.