Purpose: To investigate infant tear film secretion and protein profile, and to compare major tear proteins, such as immunoglobulin A, lactoferrin, and lysozyme, with those of adult controls. Methods: Tears were collected, with a cellulose rod, from 40 healthy infants (19 female infants and 21 male infants, gestational duration: 39.71 ± 1.27 weeks) within 48 hours of birth and 22 adults (10 female infants and 12 male infants, mean age: 24.95 ± 3.63 years). A second collection was obtained from 14 of the infants (8 female infants and 6 male infants, postnatal age: 7.76 ± 6.14 weeks). The tear volume was measured, and protein in the samples was analyzed by Bradford assay and gel electrophoresis. Results: Median tear volume (interquartile range) was 0.5μL (0.6-2 μL) for newborn infants, 2.5μL (1.4-7.75 μL) for these infants at an older age, and 6μL (2.73-12.75 μL) in adults (P < 0.001, Kruskall-Wallis test). Immunoglobulin A concentration was significantly lower in newborns (P < 0.001, analysis of variance). Lipocalin was present in 36% of the newborn tear samples, whereas serum albumin was found in 86%. Mean protein concentration (μg/μL ± SD) was 10.95 ± 5.51 in the newborns, 12.93 ± 3.99 in the older infants, and 13.04 ± 3.46 in the adults (P > 0.5, analysis of variance). Conclusions: This is the first study reporting an investigation of unstimulated infant tears, using a noninvasive collection method. Tear protein content demonstrated that the infant tear film is different to that in adults. Copyright © 2011 by Lippincott Williams & Wilkins.