A protocol for quantitative assay of several proteins in small tear volumes is described. Assays included sandwich ELISA's for IgM, IgA, secretory IgA, IgG, lactoferrin and“serum”albumin, and kinetic assays for lysozyme and peroxidase. All assays were applied to whole stimulated tears and size exclusion (SE) HPLC-separated fractions of stimulated tears. Comparison of results revealed several sources of error in whole tear assays. Tear IgG and albumin levels were considerably lower than reported by others. IgM was routinely detected at very low levels. Secretory IgA levels greatly exceeded“serum”IgA. SE-HPLC-isolated tear specific prealbumin was separated into five sub-fractions by anion exchange HPLC. Due to its ability to identify proteins in very small tear volumes, the protocol will be most useful for assay of non-stimulated tears.