Dried blood spots (DBSs) have recently gained attention as a practical alternative to conventional blood collection, offering advantages such as simplicity, minimal invasiveness, and user-friendliness. Their robustness and ease of handling have expanded their applications in both clinical and research contexts, particularly in mass spectrometry-based metabolomics and lipidomics. In this work, we optimized and evaluated extraction parameters, including solvent composition, extraction workflow, and analyte stability, to perform combined metabolomics and lipidomics analyses from a single DBS sample using three commercial microsampling devices: Capitainer, Whatman, and Telimmune. Five extraction solutions, selected from those commonly used for metabolite and lipid extraction, were tested to maximize recovery of both polar metabolites and lipids, and short-term stability of analytes at ambient temperature was assessed for up to five days. Among the solvents investigated, pure methanol emerged as the optimal single-solvent condition, providing the best compromise for co-extraction of both metabolite classes. In addition, a two-step protocol combining methanol followed by aqueous extraction significantly improved polar metabolite recovery and reproducibility, particularly in Capitainer and Whatman devices. Stability studies showed that Capitainer preserved metabolite and lipid integrity for up to six days at room temperature, whereas significant variations appeared after three days in Whatman and Telimmune devices, indicating the necessity of cold-chain storage for these platforms. In conclusion, this study identifies methanol-based extraction as the most effective approach for integrated metabolomics and lipidomics analyses from a single spot, and highlights the importance of considering device stability to ensure reliable and reproducible multi-omics analyses in clinical and translational studies.