Introduction <p>Blood microsampling (BµS) devices collect less than 100 µL of blood, offering a less invasive and more cost-effective alternative to venipuncture. However, its metabolomic comparability to conventional samples remains unclear, and standardized BµS metabolomic workflows are lacking.</p> Objectives <p>This study evaluated the impact of using three BµS devices (Mitra^®<?ExcludedElement 1T?>, Capitainer^®<?ExcludedElement 3T?>, and Whatman^™ 903) on the metabolomic interpretation of human biomonitoring samples. We compared them to conventional samples (plasma and whole blood) and evaluated the interplay of different analytical conditions.</p> Methods <p>Venous blood from 10 adults (5 males, 5 females) was sampled onto the three devices. First, three agitation conditions (ultrasound, shaker, and homogenizer) were evaluated at three blood concentrations (1.5%, 5.5%, and 11%). The optimized method was then used to compare the metabolite profiles between BµS devices, whole blood, and plasma. Reverse-phase and hydrophilic-interaction chromatography, in positive and negative ionization modes, were combined for liquid chromatography–mass spectrometry (LC-MS) analysis.</p> Results <p>All agitation conditions and concentrations proved suitable for BµS untargeted metabolomics. Combining different analytical modes and fragmentation ranges proved helpful for maximizing metabolite coverage. BµS-derived metabolite profiles aligned more closely with whole blood than plasma. Some metabolites were more characteristic of a sample type, whereas others were common across sample types. All sample types enabled sex-based differentiation, with metabolites such as amino acids, lipids, and acylcarnitines driving the separation.</p> Conclusions <p>These findings enhance our understanding of BµS metabolite coverage and highlight its potential in human biomonitoring. The choice of device depends on the application and the metabolites of interest, offering flexibility for clinical use and research.</p>