An enzyme-linked immunosorbent assay (ELISA) for detection of Varicella zoster virus (VZV) antibodies in oral fluid is described. The assay was optimised and evaluated using paired serum and oral fluid from healthy adult volunteers (n = 205) and preschool children (n = 98), oral fluid samples collected for routine measles, mumps and rubella testing (n = 537) and samples from a study of atopic dermatitis (n = 252). As chickenpox is predominantly a childhood disease and most adults are immune, it was crucial to have samples from children aged 1-5 years to evaluate the assay. Mixture modelling of the oral fluid results was used to determine the optimum cut-off, sensitivity and specificity of the assay. Compared to paired sera tested by the same ELISA the sensitivity of the oral fluid assay was 93% and specificity 95.7% overall, varying slightly with age group. The assay was shown to have good potential for use in large-scale epidemiological studies.