The objective of this study was to translate the MSQOL-54 into Serbian, and investigate the validity of the translated and cross-culturally adapted inventory in Serbian MS patients. The questionnaire was validated in 200 consecutive MS patients seen between February and September 2005 at the Institute of Neurology, Clinical center of Serbia, in Belgrade. The translation followed an internationally accepted methodology. Associations between age, gender, education, marital and employment status, disease course, the expanded disability status scale (EDSS) score, and the MSQOL-54 physical and mental health composite scores were determined. Patients’ participation in the assessment was satisfactory and all scales fulfilled the usual psychometric standards. Highly significant inverse relationship was found between both composite scores and clinical characteristics of the disease, the EDSS and the disease course. Additionally, both composite scores, correlated significantly with patients’ age, education and employment status. The Serbian-translated version of this questionnaire may be useful as clinical outcome measures in patients with MS.