X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder that affects the nervous system, and the adrenal cortex. Newborn screening for X-ALD has been proposed to allow improved diagnosis along with prospective monitoring and treatment for this severe disorder. Newborn dried whole blood spot (DBS) 26:0 lysophosphatidyl choline was validated as a diagnostic marker for X-ALD and other peroxisomal disorders of peroxisomal β-oxidation.In this study, we developed a new one step extraction procedure that simultaneously extracts acyl carnitines and the lysophosphatidyl cholines from DBS. Further analysis of these metabolites has been performed by two different high throughput LC-MS/MS methods. The 26:0 lysophosphatidyl choline levels in this study were consistent with previously published values and discriminate between healthy and abnormal profiles. There is a very minor modification to the original acyl carnitine extraction procedure and our data indicates that there is no significant effect on acyl carnitine levels in DBS.Our new method potentially can be complementary to the current newborn screening panel. It successfully combines the existing method for acyl carnitine analysis and 26:0 lysophosphatidyl choline that can be applied for prospective X-ALD newborn screening. © 2011 Elsevier Inc.