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Introduction: The presence of heavy metals in the environment has been linked to female infertility. Follicular fluid (FF), which surrounds the developing oocyte in the ovary, can provide valuable insights into the content of a number of elements. However, there is a lack of comprehensive studies that analyze trace elements in FF samples using standardized protocols and methods. The objective of this study was to develop a reliable method utilizing ICP-MS/MS without sample digestion to detect 22 analytes in FF samples. Methods: Four FF samples (n = 4) were obtained from patients undergoing in vitro fertilization (IVF) treatments. These were adequately aliquoted and processed. They were subsequently analyzed via ICP-MS/MS, both in He and no-gas mode. Finally, the data of interest were compiled in a database. Results: Analyte recovery ranged from 70% to 130%, with better results observed in no-gas mode compared to He mode. Among the 22 elements analyzed, only 9Be, 140Ce, 111Cd, 139La, 208Pb, and 238U were not detected. Minor concentrations were observed for 137Ba, 209Bi, 59Co, 55Mn, 95Mo, 60Ni, 121Sb, 118Sn, 205Tl, and 51V, while intermediate concentrations were found for 7Li and 88Sr. Major concentrations were identified for 52Cr, 85Rb, 47Ti, and 66Zn. Conclusions: A new procedure was developed in this study, with several advantages. One of them is the good results in terms of the recovery of a wide variety of elements using ICP-MS/MS in the poorly studied biological matrix that is follicular fluid. Another advantage is the low amount of sample required to perform the analysis and the development of a green method where the sample is not digested and not diluted, which allowed us to avoid the excessive dilution of the concentration of analytes. Moreover, the developed method provides a novel approach to diagnose and predict health risks in women with gynecological pathologies and to assess their overall health, including reproductive health.