Methods of treating dry eye syndrome (DES) with an effective amount of thymosin beta 4 (Tβ4), Tβ4 fragments, Tβ4 isoforms, Tβ4 derivatives, peptide agents including amino acid sequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO:2], or variants thereof are provided. The presently disclosed subject matter provides methods of increasing tear volume, increasing tear film stability, decreasing ocular surface damage, and decreasing ocular discomfort by delivering compositions of thymosin beta 4 or fragments thereof to subjects in need.