BACKGROUND AND PURPOSE: Lesion volume change (LVC) assessment is essential in monitoring MS progression. LVC is usually measured by independently segmenting serial MR imaging examinations. Subtraction imaging has been proposed for improved visualization and characterization of lesion change. We compare segmentation of subtraction images (SSEG) with serial single time-point conventional segmentation (CSEG) by assessing the LVC relationship to brain atrophy and disease duration, as well as scan-rescan reproducibility and annual rates of lesion accrual. MATERIALS AND METHODS: Pairs of scans were acquired 1.5 to 4.7 years apart in 21 patients with multiple sclerosis (MS). Scan-rescan MR images were acquired within 30 minutes in 10 patients with MS. LVC was measured with CSEG and SSEG after coregistration and normalization. Coefficient of variation (COV) and Bland-Altman analyses estimated method reproducibility. Spearman rank correlations probed associations between LVC and other measures.