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This paper describes and compares novel approaches toin vivo3D measurement of brain surface folding in clinically acquired neonatal MR image data, which allows regional folding evaluation. Most of the current measures of folding are not independent of the area of the surface they are derived from. Therefore, applying them to whole-brain surfaces or subregions of different sizes results in differences which may or may not reflect true differences in folding. We address this problem by proposing new measures to quantify gyrification and two approaches to normalize previously defined measures. The method was applied to twelve premature infants (age 28–37 weeks) from which cerebrospinal fluid/gray matter and gray matter/white matter interface surfaces were extracted. Experimental results show that previous folding measures are sensitive to the area of the surface of analysis and that the area-independent measures proposed here provide significant improvements. Such a system provides a tool that facilitates the study of structural development in the neonatal brain within specific functional subregions, which may be critical in identifying later neurological impairment.