Bee pollen collected by honeybees () is one of the bee products, and it is as valuable as honey, propolis, royal jelly, or beebread. Its quality varies according to its geographic location or plant sources. This study aimed to apply rapid, simple, and accurate analytical methods such as attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and high-performance liquid chromatography (HPLC) along with chemometrics analysis to construct a model aimed at discriminating between different pollen samples. In total, 33 samples were collected and analyzed using principal component analysis (PCA), hierarchical clustering analysis (HCA), and partial least squares regression (PLS) to assess the differences and similarities between them. The PCA score plot based on both HPLC and ATR-FTIR revealed the same discriminatory pattern, and the samples were divided into four major classes depending on their total content of polyphenols. The results revealed that spectral data obtained from ATR-FTIR acquired in the region (4000-500 cm) were further subjected to a standard normal variable (SNV) method that removes scattering effects from spectra. However, PCA, HCA, and PLS showed that the best PLS model was obtained with a regression coefficient ( ) of 0.9001, root-mean-square estimation error (RMSEE) of 0.0304, and root-mean-squared error cross-validation (RMSEcv) of 0.036. Discrimination between the three species has also been possible by combining the pre-processed ATR-FTIR spectra with PCA and PLS. Additionally, the HPLC chromatograms after pre-treatment (SNV) were subjected to unsupervised analysis (PCA-HCA) and supervised analysis (PLS). The PLS model confers good results by factors ( = 0.98, RMSEE = 8.22, and RMSEcv = 27.86). Prospects for devising bee pollen quality assessment methods include utilizing ATR-FTIR and HPLC in combination with multivariate methods for rapid authentication of the geographic location or plant sources of bee pollen.