Computational and experimental methods were applied to investigate the self-assembly and gelation of C13-dipeptides. A modified aggregation propensity (APS) was introduced to correlate the effects of side chains of amino acids on the tendency to aggregate. From the experimental results, the ranges of 0.156 < APS < 0.250 seemed to be a proper region for the C13-dipeptides to form hydrogels, while other molecules with higher or lower APS were insoluble or dissociated. As observed from molecular dynamics simulations, the C13-dipeptides first formed small aggregates through hydrophobic interactions and then rearranged through electrostatic attractions and hydrogen bonds for self-assembly. The C13-dipeptides tended to be antiparallel packed, as shown by hydrogen bonding analyses. Experimental observations and analyses on the structures of C13-dipeptide hydrogels matched the computational conclusions very well. From the five selected gelators, i.e., C13-GW, C13-VY, and C13-WT, strong π-π stacking was observed. For C13-WS, strong hydrogen bonding was found, and in C13-WY, both strong π-π interactions and hydrogen bonds were found. It takes around 90 min or longer for C13-dipeptides to form hydrogels, and those formed by C13-WY and C13-WS had weak water holding capacities, which might be due to strong intermolecular hydrogen bonding. From rheological studies, the C13-dipeptides formed strong chemical gels that were stabilized by strong interactions between the molecular aggregates. These gelators exhibit the potentials to be environmentally friendly substitutes for the common functionalized peptide gelators.