The presence of acanthocytosis in peripheral blood smears remains the hallmark of the clinical diagnosis of most neuroacanthocytosis syndromes, such as chorea-acanthocytosis (ChAc) and McLeod syndrome. Genetic analyses and/or specific laboratory tests are available only for a minority of these disorders. Testing for acanthocytosis is hampered by the lack of data on normal amounts of acanthocytes assessed by a standardized method. We report a prospective reader-blinded study designed to establish control values for abnormally shaped erythrocytes in healthy volunteers and patients with movement disorders (MDs) using light microscopic assessment of erythrocyte morphology in standard EDTA and isotonically diluted blood samples. We investigated a total of 100 patients fulfilling clinical criteria of specific MDs, 31 patients with MDs not matching any clinical criteria, and 37 healthy controls. In patients with diagnosed MDs and healthy controls, acanthocytes in dry blood smears were significantly more frequent following isotonic dilution compared with standard EDTA blood. In unfixed wet blood preparations of both EDTA blood and isotonically diluted blood, acanthocyte levels were significantly higher than in standard dry blood smear preparations. There were no statistical differences of acanthocyte levels in all test conditions between diagnosed MDs and healthy volunteers. There was no significant correlation of acanthocyte levels in all blood samples and preparations with age, sex or diagnosis. Thus, normal values were defined as the 99th percentile of combined results of the two groups of volunteers. Diluted blood combined with wet blood preparation showed high specificity (0.98) and the highest sensitivity of all test procedures (all genetically confirmed ChAc patients were detected). The reported method is cheap, readily available, and provides high specificity and sensitivity in respect to clinically relevant acanthocytosis. The use of isotonically diluted blood samples combined with unfixed wet blood preparation with a normal range of <6.3% of total erythrocytes is recommended to search for significant acanthocytosis in movement disorders.