The use of metabolomics profiling to understand the metabolism under different physiological states has increased in recent years, which created the need for robust analytical platforms. Here, we present a validated method for targeted and semiquantitative analysis of 102 polar metabolites that cover major metabolic pathways from 24 classes in a single 17.5-min assay. The method has been optimized for a wide range of biological matrices from various organisms, and involves automated sample preparation and data processing using an inhouse developed R-package. To ensure reliability, the method was validated for accuracy, precision, selectivity, specificity, linearity, recovery, and stability according to European Medicines Agency guidelines. We demonstrated an excellent repeatability of retention times (CV < 4%), calibration curves (R2 ≥ 0.980) in their respective wide dynamic concentration ranges (CV < 3%), and concentrations (CV < 25%) of quality control samples interspersed within 25 batches analyzed over a period of one year. The robustness was demonstrated through a high correlation between metabolite concentrations measured using our method and the NIST reference values (R2 = 0.967), including cross-platform comparability against the BIOCRATES AbsoluteIDQp180 kit (R2 = 0.975) and NMR analyses (R2 = 0.884). We have shown that our method can be successfully applied in many biomedical research fields and clinical trials, including epidemiological studies for biomarker discovery. In summary, a thorough validation demonstrated that our method is reproducible, robust, reliable, and suitable for metabolomics studies.