AbstractBackgroundCancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis.AimsThis study aimed to evaluate the changes in the amino acid profile in MCF‐7 and MDA‐MB‐231 cells during spheroid formation to discover the specific metabolic properties in CSCs.MethodsMCF‐7 and MDA‐MB‐231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high‐performance liquid chromatography (HPLC).ResultsThe results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF‐7 and MDA‐MB‐231 spheroids (histidine and serine, respectively).ConclusionOur results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation.