This file is including the results of in vitro phosphatidylglucoside (PtdGlc) biosynthesis assay. We incubated saturated phosphatidic acid (PA, 18:0/20:0) and UDP-glucose with buffer, flag-conjugated UGGT1 or flag-conjugated UGGT2. PtdGlc (18:0/20:0) formation was monitored by reversed phase LC-MS/MS in negative ion mode with multiple reaction monitoring utilising four precursor-product ion pairs simultaneously at the following settings: m/z 893.5→283.1 (CE: 66.0); m/z 893.5→311.2 (CE: 56.0); m/z 893.5→419.1 (CE: 37.0); and m/z 893.5→79.0 (CE: 97.0). We also incubated unstaurated PA (18:1/18:1) with buffer or flag-conjugated UGGT2. The unsaturated glucosylated-PA (18:1/18:1) was detected with multiple reaction monitoring utilising three precursor-product ion pairs simultaneously at the following settings: m/z 861.5→281.1 (CE: 66.0); m/z 861.5→417.1 (CE: 37.0); and m/z 861.5→79.0 (CE: 97.0). In addition, we performed PtdGlc biosynthesis assays with flag-conjugated UGGT2 in the presence of denatured Urea-denatured thyroglobulin and saturated PA (18:0/20:0).