Candida glabrata is the second most reported Candida species causing candidiasis next only to Candida albicans. Biofilm formation by C. glabrata accelerates pathogenicity, so understanding conditions for biofilm formation in relevant polystyrene polymers is essential. Determining optimum conditions for biofilm formation using one variable at a time (OVAT) is laborious and inaccurate. Therefore, mathematical modelling for design of experiments to optimize conditions for biofilm formation in Candida species was undertaken. In this study, a simple and robust statistical method, response surface methodology (RSM) was used to optimize independent variables like temperature, incubation period, media pH, shaker speed and inoculum density. Biofilm forming conditions were optimized and compared for two important local clinical isolates and one standard culture on 96 well polystyrene microtiter plates. Biofilm was quantified by different methods, like XTT for cell viability, crystal violet for biofilm, calcofluor white, and wet and dry weight measurements for cell mass. Quantification of cell viability and biofilm indicated heterogeneity among the three cultures. The results revealed an important finding that foetal bovine serum (FBS) does not significantly affect biofilm formation in vitro. This simple high throughput method for optimization and quantification of Candida biofilm has relevance for applications in rapid screening of anti-Candida biologics and therapeutic solutions while advancing control measures in polystyrene carriers in hospital settings.