Time-resolved optical waveguide absorption spectroscopy (OWAS) makes use of an evanescent field to detect the polarized absorption spectra of sub-monomolecular adlayers. This technique is suitable for the investigation of kinetics at the solid–liquid interface of dyes, pigments, fluorescent molecules, quantum dots, metallic nanoparticles, and proteins with chromophores. In this work, we demonstrate for the first time the application of non-negative matrix factorization (NMF) to the analysis of time-resolved OWAS data, because NMF prevents the negative factors from occurring, avoids contradicting physical reality, and makes factors more easily interpretable. Meanwhile, principal component analysis (PCA) is researched for the comparison of NMF. Two-factor loadings and scores derived by PCA and NMF are consistent with the results analyzed by the function of time/wavelength-absorbance. Moreover, NMF avoids contradicting physical reality, and makes factors more easily interpretable. We believe that NMF will provide a valuable analysis route to allow processing of increasingly large and complex data sets.