Aim To investigate the ocular surface inflammatory response to chronic topical treatments in patients with glaucoma by measuring the cytokine level in tears using multiplex bead analysis. Methods Tear samples were collected from 21 patients with glaucoma and 12 healthy volunteers. Tears were analysed for the presence of 17 cytokines: interleukin (IL)1β, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL13, IL17, granulocyte‐colony stimulating factor, granulocyte‐macrophage stimulating factor, interferon (INF)γ, monocyte chemotactic protein (MCP)1, macrophage inflammatory protein 1β and tumour necrosis factor (TNF)α. The cytokines in each sample of tears were measured using multiplex bead analysis with microspheres as solid support for immunoassays. Results In the tears of treated patients, proinflammatory cytokines (IL1β, IL6, IL12, TNFα) were significantly increased compared with controls. T helper (Th)1 (INFγ, IL2) and Th2 (IL5, IL10, IL4) type cytokines were also significantly higher (p<0.05); however, the most marked increase was observed with Th1 cytokines. The expression of chemokine IL8 and MCP1 was also increased in the treated group. Conclusion This study shows that pro‐inflammatory cytokine secretion by conjunctival cells is increased in response to topical treatments for glaucoma. The characterisation of cytokines in tears was previously limited by the small volume attainable, a limitation that has been overcome by multiplex analysis.