This study developed and validated a robust UPLC-MS/MS method for quantifying cyclic glycine–proline (cGP) in mangrove-derived Penicillium and Aspergillus strains. The method demonstrated excellent linearity, precision, and recovery, with detection limits as low as 4.8 ng/mL. Penicillium pedernalense extract achieved a cGP content of 67.45 ± 1.11 ng/mL, with a corresponding fermentation yield of 29.31 ± 0.61 mg/L. This surpassed Penicillium steckii, which reached a content of 31.71 ± 0.31 ng/mL, with a yield of 8.51 ± 0.15 mg/L. This quantitative approach for metabolite analysis provides a viable method for screening these fungal strains, highlighting their potential for sustainable production of cyclic glycine–proline (cGP).