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Breast cancer (BC) is among the most commonly diagnosed cancers. Besides mammography, breast ultrasonography and the routinely monitored protein markers, the variations of small molecular metabolites in blood may be of great diagnostic value. This study aimed to quantify specific metabolite markers with potential application in BC detection. The study enrolled 50 participants, 25 BC patients and 25 healthy controls (CTRL). Dried blood spots (DBS) were utilized as biological media and were quantified via a simplified liquid chromatography tandem mass spectrometry (LC-MS/MS) method, used in expanded newborn screening. The targeted metabolomic analysis included 12 amino acids and 32 acylcarnitines. Statistical analysis revealed a significant variation of metabolic profiles between BC patients and CTRL. Among the 44 metabolites, 18 acylcarnitines and 10 amino acids remained significant after Bonferroni correction, showing increase or decrease and enabled classification of BC patients and CTRL. The well-established LC-MS/MS protocol could provide results within few minutes. Therefore, the combination of an easy-to-handle material-DBS and LC-MS/MS protocol could facilitate BC screening/diagnosis and in the next step applied to other cancer patients, as well.