We present a comparative study of the time-of-flight-secondary ion mass spectrometry (ToF-SIMS), Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS) techniques on a mixed self-assembled monolayer (SAM) of biotinylated polyethylene glycol alkane thiol (BPT) and 11-mercaptoundecanol (MUD) as a model system. This mixed SAM was employed to maximize specific binding between the biotin groups in the mixed SAM and the streptavidin, while minimizing nonspecific adsorption of the streptavidin. For a better quantitative analysis of our mixed SAM by using ToF-SIMS, we performed a principal component analyses (PCA) with various peak sets and found that the negative ions of the MUD complementary peak set showed good correlation between the XPS and FT-IR quantification results and the scores for principal component 1 from a PCA of the ToF-SIMS data. These findings show that ToF-SIMS analysis along with PCA would make a good surface analysis technique for identifying intact molecules of adsorbates and for quantifying at once each adsorbate in a mixed SAM.