This article provides a detailed dataset of human tear fluid proteins. Samples were fractionated by sodium dodecyl sulfate (SDS) gel electrophoresis resulting in 48 fractions that were spiked with an indexed retention time (iRT) peptide standard. These data are based on a data-dependent acquisition (DDA) mass spectrometric approach and can be used for example as a spectral library for tear fluid proteome analysis by data-independent acquisition (DIA). Moreover, the provided data set can be used with optimized HPLC as well as mass spectrometric settings for proteins/peptides of interest. Besides these aspects, this dataset can serve as a protein overview for gene ontology enrichment analysis as well as for modeling and benchmarking of multiple signaling pathways associated with the ocular surface in healthy or disease stages.