In mass spectrometry, reliable quantification requires correction for variations in ionization efficiency between samples. The preferred method is the addition of a stable isotope-labeled internal standard (SIL-IS). In targeted metabolomics, a dedicated SIL-IS for each metabolite of interest may not always be realized due to high cost or limited availability. We recently completed the analysis of more than 70 biomarkers, each with a matching SIL-IS, across four mass spectrometry-based platforms (one GC–MS/MS and three LC–MS/MS). Using data from calibrator and quality control samples added to 60 96-well trays (analytical runs), we calculated analytical precision (CV) retrospectively. The use of integrated peak areas for all metabolites and internal standards allowed us to calculate precision for all matching analyte (A)/SIL-IS (IS) pairs as well as for all nonmatching A/IS pairs within each platform (total n = 1442). The median between-run precision for matching A/IS across the four platforms was 2.7–5.9%. The median CV for nonmatching A/IS (corresponding to pairing analytes with a non-SIL-IS) was 2.9–10.7 percentage points higher. Across all platforms, CVs for nonmatching A/IS increased with increasing difference in retention time (Spearman’s rho of 0.17–0.93). The CV difference for nonmatching vs matching A/IS was often, but not always, smaller when analytes and internal standards were close structural analogs.