We previously proposed a new method for exploring functional peptides using both spot-synthesis peptide libraries and principal component analysis (PCA). Here, we applied these methods to determine if high-binding 6-mer peptides can be used on bile acid for the inhibition of intestinal cholesterol absorption. We used a binding assay of 512 basal 4-mer peptides to bile acid, and from these selected high-binding and low-binding peptides. PCA was performed on data from both these binding groups and many physicochemical variables of the 512 peptides tested, and then through this, the variables were reduced to two principal components (PCs). The peptides were plotted on the PCA chart, and we identified distinct clusters of high- and low-binding regions. These PCA regions were applied to 6-mer random peptides, and we identified 6-mer peptides with high and low binding capacity to bile acid. We confirmed that the average fluorescence intensity of high-binding peptides was 3.0-fold higher than that of low-binding peptides. We succeeded in identifying 6-mer peptides with high and low-binding affinity based on the PCA analysis of 4-mer peptides. These results were compared and discussed with regard to those acquired by our previous computational analysis based on neural networks. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.