Little information is available in the literature regarding trace element analysis of human follicular fluid. Most peer-reviewed publications offer few details about sample preparation, method development, or method validation. We have attempted to fill this gap in current knowledge by providing comprehensive details of our method development and validation process for the measurement of 24 trace elements (As, Ba, Be, Cd, Co, Cr, Cs, Cu, Hg, Mo, Mg, Mn, Pb, Pt, Rb, Se, Sb, Sn, Sr, Tl, U, V, W, Zn) in follicular fluid. A number of different sample preparation procedures were explored, with a goal of optimizing method detection limits (MDLs), for both quadrupole-inductively coupled plasma-mass spectrometry (Q-ICP-MS) and sector field (SF)-ICP-MS. Human follicular fluid samples (n = 72) were obtained from 64 women undergoing in vitro fertilization (IVF) treatment, and were successfully analyzed for 24 trace elements using SF-ICP-MS.