AbstractOvarian reserve (OR) and fertility are critical in women's healthcare. Clinical methods for encoding OR and fertility rely on the combination of tests, which cannot serve as a multi‐functional platform with limited information from specific biofluids. Herein, metabolic fingerprinting of follicular fluid (MFFF) from follicles is performed, using particle‐assisted laser desorption/ionization mass spectrometry (PALDI‐MS) to encode OR and fertility. PALDI‐MS allows efficient MFFF, showing fast speed (≈30 s), high sensitivity (≈60 fmol), and desirable reproducibility (coefficients of variation <15%). Further, machine learning of MFFF is applied to diagnose diminished OR (area under the curve of 0.929) and identify high‐quality oocytes/embryos (p < 0.05) by a single PALDI‐MS test. Meanwhile, metabolic biomarkers from MFFF are identified, which also determine oocyte/embryo quality (p < 0.05) from the sampling follicles toward fertility prediction in clinics. This approach offers a powerful platform in women's healthcare, not limited to OR and fertility.