Context <p>Hydroxylated derivatives of progesterone are biologically relevant compounds obtained through regio- and stereoselective biotransformations, but determining their stereochemical configuration is challenging due to subtle NMR chemical shift differences. In this study, monohydroxylated (prog-15-OH) and dihydroxylated (prog-7,15-di-OH) progesterone derivatives produced by <i>Penicillium oxalicum</i> CBMAI 1996 were analyzed. Quantum NMR calculations combined with statistical validation allowed the unambiguous identification of the 15β-OH and 7β,15β-di-OH isomers, with DP4+ probabilities of 100% and 99.8%, respectively. These findings confirm the stereochemical outcome of enzymatic hydroxylation and demonstrate the reliability of integrating computational NMR with experimental spectroscopy for resolving stereochemical ambiguities in complex steroidal frameworks.</p> Methods <p>Conformational searches were performed using the MMFF94 force field, followed by DFT geometry optimizations at the B3LYP/6-311G(2d,p) level with IEFPCM (chloroform). NMR shielding tensors were computed by the GIAO method at the mPW1PW91/6-311G(d,p) level with PCM (chloroform), and chemical shifts were referenced to TMS. Experimental and calculated ¹H/¹³C shifts were compared using correlation coefficients (<i>R</i>²), mean absolute deviation (MAD), root mean square deviation (RMSD), and DP4+ probability analysis. All calculations were performed with Gaussian 16.</p>