Twenty-four Alternaria strains were isolated from barley grain samples. These strains were screened for the production of mycotoxins on rice medium using thin layer chromatography. All 24 strains produced at least one of the five mycotoxins (ALT, AOH, ATX-I, AME, and TeA). Three representative strains, namely EML-BLDF1-4, EML-BLDF1-14, and EML-BLDF1-18, were further analyzed using a new LC–MS/MS-based mycotoxin quantification method. This method was used to detect and quantify Alternaria mycotoxins. We used positive ion electrospray mass spectrometry with multiple reaction mode (MRM) for the simultaneous quantification of various Alternaria mycotoxins produced by these strains. Five Alternaria toxins (ALT, ATX-I, AOH, AME, and TeA) were detected and quantified. Sample preparation included methanol extraction, concentration, and injection into LC–MS/MS. Limit of detection ranged from 0.13 to 4 μg/mL and limit of quantification ranged from 0.25 to 8 μg/mL
