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Abstractl-homoarginine (hARG) is involved in nitric oxide biosynthesis, but its role and concentration in preeclampsia (PE) have not been fully revealed. The purpose of this study was to develop and validate a feasible clinical assay to quantify serum hARG, arginine (ARG), asymmetric (ADMA) and symmetric dimethylarginines (SDMA) levels by LC–MS/MS and investigate their differences at different stages of pregnancy with or without preeclampsia. Serum samples were collected from 84 pregnant women without complications (controls), 84 with mild preeclampsia (MPE), and 81 with severe preeclampsia (SPE) at various gestation stages (before the 20th week, during the 20th–28th week or after the 28th week of gestation). No significant difference in ARG levels was observed between PE and controls at any stage (P > 0.05). The serum hARG levels and hARG/ADMA ratios of MPE before the 20th week were higher than those of controls (P < 0.001). ADMA levels of MPE were higher than those of controls during the 20th–28th week (P < 0.01). SDMA levels of SPE were higher than those of MPE (P < 0.01) and controls (P < 0.05) after the 28th week. Elevated serum hARG before the 20th week was identified as an independent predictor for PE (OR = 1.478, 95% CI 1.120–1.950). ROC curve analysis showed serum hARG before the 20th week had a good potential to predict MPE (AUC = 0.875, 95% CI 0.759–0.948). In conclusion, our study indicated that elevated serum hARG and dimethylarginine levels detected by LC–MS/MS might serve as potential biomarkers for the early prediction of PE.