The proteins and peptides in tears play an important role in preserving the integrity and stability of the ocular surface. Proteomic analysis of tear films will enable us to detect early biological markers of eye diseases, however, it is often hampered by the small amount of tear volume and the low protein concentration. Here we adopted magnetic beadbased purification (ClinProt system) followed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to profile human tear proteins. Basal and reflex tear fluids were collected from normal healthy volunteers using glass microcapillary tubes. Reversed phase (C8) and weak cation exchange (WCX) magnetic beads were applied to obtain multiple components detected as clear signals. Principal component analysis showed a clear differentiation between basal and reflex tears. Among the key alterations, two markedly increased peaks in the reflex tear fluids at m/z 2422.12 and m/z 2721.29 were subsequently analyzed by tandem MS analysis and their source to be proline-rich protein 4 (PRP4). We conclude that magnetic bead-based separation combined with MALDI-TOF-MS (ClinProt MALDI-TOF) appears to be ideally suited for the first-line screening of peptides and proteins in tears.