Homochirality is one of the mysterious chemical benchmarks that is relevant to the origin of life. Biomacromolecules, constructed from the chiral building blocks, are mostly from exclusively either L- or D- structures, and the vast majority of the reactions in aqueous media are fine-tuned by chiral selectivity. However, although characterized by sum-frequency generation (SFG) spectroscopy and other techniques, whether and how the chiral biomolecules affect their hydration shells, through which intermolecular forces of such molecules are transmitted, are far from clear. Yet, the effect of perturbing the homochirality of biomacromolecules on their self-assembly is thus ambiguous. Here, I propose to use surface-anchored chiral short peptide molecules with a well-defined primary structure as a model. The structure and dynamics of the hydration shell of this peptide will be studied by chiral heterodyne detected sum-frequency (HD-SFG) spectroscopy, a nonlinear optical technique that provides information on hydration shells and their chirality. Using the solid phase peptide synthesis technique, chiral inversion will be induced at the level of a single monomer, blocks, and mixture of enantiomers. The self-assembly of peptide molecules will be characterized by atomic force microscopy for their morphology and SFG for their secondary structure. By quantitively comparing the spectrum of enantiomers, the dipole orientation of hydration water and their HB-network relaxation will be characterized using the O-H stretching band, which reflects thermodynamics and kinetics properties. Furthermore, the chiral inversion effect on self-assembly will be measured in the peptide’s amide I band, which reflects their packing. These results will shed light on chiral-induced water superstructure and self-assembly modulation, which is crucial for understanding the biological enrichment of homochirality and for anticancer/antiviral agent design.