Toxic metals found in the environment have been linked to female infertility and gynecological illnesses. Reliable analytical methods, such as inductively coupled plasma tandem mass spectrometry (ICP-MS/MS), are necessary to determine the elemental composition of biological samples. Currently, the multielemental profile of peritoneal fluid (PF) samples has not yet been established. Due to the complexity of the PF matrix, an ICP-MS/MS-based method has been optimized to mitigate matrix effects and spectral interferences. A dilution factor of 1:4 was the best option to mitigate matrix effects while keeping sensitivity at an appropriate level. A collision gas (He) was useful to lower the extent of spectral interferences for56Fe,52Cr,63Cu, and68Zn. An intermediate validation test was performed to evaluate accuracy, achieving recoveries ranging from 90 to 110%. The method was validated in terms of intermediate precision, reproducibility, and trueness, with an expanded uncertainty lower than 15%. Afterward, it was applied to perform the multielemental analysis of 20 PF samples. The concentrations for major analytes were up to 151 µg L−1. Meanwhile,209Bi,111Cd,52Cr,55Mn,95Mo,60Ni,208Pb,118Sn, and51V were present at concentrations included within the 1–10 µg L−1range, while59Co and139La were found at concentrations below 1 µg L−1.