Hydrogels is a class of material that composed of heavily hydrated network. It has been studied and used in various biomedical applications including drug delivery, cell therapy and tissue engineering. In our lab, we are interested in generating an acidic peptide-based hydrogel for cellular delivery and tissue engineering. Here, β-hairpin peptides capable of folding and self-assembly affording self-supportive hydrogels were rationally designed. Then, acidic amino acids with different side-chain length were incorporated into each peptide to investigate the folding, self-assembling and subsequently rheological behavior of the hydrogels. The resulted peptides are VX, VE and VD, composed of aminoadipic acid, glutamic acid and aspartic acid as one of the building blocks respectively. Each peptide undergoes gelation in response to changes in environmental pH and temperature. The side-chain length of amino acids dictates the conditions at which folding and self-assembly is permitted. For examples, at 50oC pH 7.0, VX folds and self-assemble affording self-supporting hydrogel while VD remains unfold as shown by circular dichroism spectroscopy and oscillatory rheology. All VX, VE and VD display similar network morphologies as revealed by transmission spectroscopy even though the building blocks are different. To generate acidic hydrogels that can form under physiological conditions, VE3 and VEQ1 were then designed by controlling peptides' charge state. Both gels can be used to directly encapsulate cells providing homogeneous cellular distribution. The shear-thinning and recovery property as well as the cytocompatility of the VE3 and VEQ1 gels make them promising cell carriers and scaffolds for tissue engineering applications.