X-linked adrenoleukodystrophy (X-ALD) is a rare inherited metabolic disease that results in the accumulation of very long chain fatty acids (VLCFA) in plasma and all tissues. Recent studies regarding cerebral X-ALD (CALD) treatment emphasize the importance of its early diagnosis. 26:0 lysophosphatidylcholine (LysoPC) is a sensitive biomarker for newborn screening of X-ALD, while its application for Japanese DBS is unclear. Therefore, we evaluated the feasibility of 20:0 LysoPC and 24:0 LysoPC along with 26:0 LysoPC for diagnosing X-ALD in a cohort of newborns (n = 604), healthy adults (n = 50) and patients (n = 4). Results indicated that 26:0 LysoPC had strong significance for discrimination of patients by the amounts of 2.0 to 4.0 and 0.1 to 1.9 pmol/punch for patients and newborns/healthy adults, respectively. Based on these values, we recommend that further diagnostic confirmation is essential if the amount of 26:0 LysoPC in DBS is above 1.7 pmol/punch.