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X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder that affects the nervous system and adrenal glands. C26:0-lysophosphatidylcholine (LPC) is a biomarker for X-ALD and is elevated in effected newborns. A flow injection analysis-tandem mass spectrometry (MS/MS) method is developed for the analysis of LPC along with amino acids (AA), succinylacetone (SA) and carnitines (AC) from dried bloodspots (DBS) in a single assay. Two sets of AA/AC/SA DBS controls (five levels each), one with C20:0-,C22:0-,C24:0-and C26:0-LPC and the other without, are prepared. The extraction of LPC from a single DBS (3.2 mm) is optimized using several different extraction solvent mixtures of ACN-methanol-water and ACN-EA-water. Extraction time (30-60 min) and temperature (27°C-45°C) are optimized to improve recovery. The internal standard used for the analysis of LPC is 2H4-C26:0-LPC. The analysis time is 1.5 minutes. The MRM transitions monitored for quantitation of C26:0-LPC is m/z 636 to 104 for the analyte and m/z 640 to 104 for the internal standard. The recovery of C26:0-LPC ranges from 76.0% to 99.0%. No significant difference is observed in the recovery of AA, AC and SA between each levels of LPC-spiked and nonspiked controls. The coefficient of variation (CV) ranges for the LPC-spiked controls are 4.28-7.44, 4.50-7.4, 16.2, and 5.16 for AA, AC, 26:0-LPC and SA respectively and comparable with controls not spiked with LPCs. More than 1,000 newborn specimens were analyzed using the current nonderivatized MS/MS method and this new method developed for LPC/AA/AC/SA. The data obtained by both methods are comparable for all analytes. This new and simplified MS/MS method for the analysis of LPC along with AA, AC and SA in a single assay will significantly reduce the cost and time compared to the currently available methods for the detection of X-ALD along with other inborn errors of metabolism in newborns.