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This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed-phase solid-phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic-lipophilic balance, and mixed-mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed-mode anion exchange (similar to 50%) cartridge, compared to other sorbents from 100 L plasma sample. Complete analytical separation of carglumic acid and carglumic acid-13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 x 4.6 mm, 5 mu m) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00-6000 ng/mL (r(2) 0.9987). Matrix effect was evaluated in terms of IS-normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra- and inter-batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject. Copyright (c) 2015 John Wiley & Sons, Ltd.