The fragmentation mechanisms of progesterone have been studied by triple quadrupole tandem mass spectrometry (MSMS) and density functional theory (DFT). Mechanisms leading to major product ions are proposed. The data suggest that progesterone fragments preferentially via hydrogen and other rearrangements lead to neutral losses. These fragmentations are quite complex and are preceded by σ-bond cleavages in most cases. Four major pathways for progesterone fragmentation are proposed involving: (1) cleavage of ring B at C9-C10, (2) cleavage of C6-C7 bond in ring B through m/z 191, (3) two types of cleavages of ring D, and (4) ketene elimination in ring A. Pathways (1)-(3) proceed via charge-remote fragmentations while pathway (4) proceeds via charge-site initiated mechanism. The geometry of product ions in these pathways were optimized using DFT at the B3LYP/6-311G(d,p) level of theory from which the free energies of the pathways were calculated. The effect that the choice of basis sets and density functionals has on the results was tested by performing additional calculations using B3LYP/6-31G(d) and B3PW91/6-311G(d,p).