Objectives: The purpose of this study was to determine the prevalence of multiple sclerosis (MS) in Australia in 2010 using a novel method based on Australia-wide prescription data for MS-specific disease modifying agents. The results obtained were validated against two other prevalence estimates. Methods: We obtained the total number of scripts for medications that were used exclusively for the treatment of MS written in Australia for the period January–December 2010. The percentage of MS patients using medications (42–55%) was taken from state-specific surveys of MS Society clients. To estimate prevalence we divided the annual number of scripts dispensed by 12 and adjusted for penetration of medications by state. Results: The prevalence of MS in Australia in 2010 calculated using the prescription method was 21,283 people (95.5/100,000). This compared to 21,200 people (95.2/100,000) obtained from the Australian Bureau of Statistics (ABS) Survey of Disability, Ageing and Carers (SDAC) survey of 2009 and 20,471 people (91.9/100,000) using MS Society client numbers. Prevalence increased with increasing latitude, with the prevalence for Tasmania over seven times that of the Northern Territory. Results were sensitive to the percentage of people with MS being treated. Conclusions: Calculation of prevalence of MS using nation-wide prescription data is a novel method that generates results similar to other potentially more resource-intensive methods.