Many low molecular weight substances in human tears, including protein and lipid species, have yet to be characterized. Some of these uncharacterized substances may well be important in the pathogenesis of ocular surface disease or in ocular discomfort. The aim of this study was to build a biochemical profile of low molecular weight species in tears, and to determine its repeatability. A total of 80 tear samples were collected from 11 subjects. Tear samples were dialysed to remove salts, added to a matrix of α-cyano-4-hydroxycinnamic acid, and analysed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Species were separated based on their mass to charge ratio (m : z). The repeatability of the appearance of the different species was analysed using logistic regression and diurnal and day-to-day repeatability were ascertained. Peptides were identified in the range of 848-3897 Da. Of these, 39 peptides were found to be present in more than 10 / 80 samples. There was no diurnal variation in the exception of peptide 1653 Da. This study has demonstrated that the majority of low molecular weight species in tears are repeatably present and do not exhibit diurnal variation. Further study aims to characterize these species and to identify changes in tear profiles between subject groups.