Quality of life (QoL) is discussed as an additional outcome measure in multiple sclerosis (MS). However, few questionnaires assessing disease specific QoL in MS have been published. On the basis of the literature and interviews with clinicians and MS patients, we have developed a disease specific QoL instrument and validated it in a broad range of patients with MS. In this study, a heterogeneous sample of n = 237 MS patients completed the newly developed Hamburg Quality of Life Questionnaire in Multiple Sclerosis (HAQUAMS, in German language) and a battery of already validated questionnaires. They further underwent neurological scoring and objective tests. By these means, we investigated its validity, appropriateness, internal consistency, and retest reliability. Internal consistency and retest coefficients were high and satisfied psychometric standards. Convergent and discriminant validity was supported by direction, magnitude and pattern of correlations with other health measures. HAQUAMS subscales and its total score distinguished between patient groups of varied disease severity, cognitive impairment, and affective symptomatology. No floor or ceiling effects were found in either of the HAQUAMS subscales. The HAQUAMS is a reliable, valid and appropriate instrument for QoL assessment in multiple sclerosis. Data of responsiveness are currently being obtained.