A lack of standardized evaluation procedures for dopamine transporter (DAT) SPECT investigations impairs both intra- and interindividual comparisons as well as multicenter trials—for example, for assessment of disease progression or the response to various drug treatments. Therefore, the aim of this study was to evaluate a novel automated method, which has been specifically developed for a standardized quantification of N-(3-fluoropropyl)-2β-carbomethoxy-3β-(4-iodophenyl)nortropane (¹²³I-FP-CIT) SPECT studies. Methods: DAT binding ratios of 155 ¹²³I-FP-CIT SPECT studies in 14 control subjects and 141 patients referred to confirm or exclude a presynaptic dopaminergic deficit were determined manually and by a fully automated technique. The latter included coregistration of patient studies to an ¹²³I-FP-CIT mean template of controls with specialized, nonrigid adjustment for variation in striatal location, followed by calculation of specific striatal DAT binding using a standardized 3-dimensional volume-of-interest (VOI) map. The map is based on a MR scan and covers the striatum (S), caudate (C), putamen (P), and an occipital reference region. The semiquantitative ratios of both methods were compared with the visual findings. Results: Excellent linear correlations were observed between manually and automatically determined results (S: r = 0.99; C: r = 0.99; P: r = 0.99; P < 0.001, respectively). Automated evaluations delivered highly reproducible and visually exact coregistrations. Individual variations in striatal anatomy (e.g., atrophy) were considered and VOI positions were corrected before quantification. Both the manual and the automated method showed identical accuracy in supporting the visual diagnoses. Conclusion: In a large patient population, excellent agreement was observed between quantitative DAT results using a time-consuming, observer-dependent, conventional manual method and the objective, automated evaluation specifically developed for a standardized evaluation of ¹²³I-FP-CIT SPECT studies. It is suggested that the novel automated technique may substantially facilitate both intra- and interindividual comparisons as well as multicenter trials.