Mitochondrial permeability transition pore (MPTP) dysregulation can be correlated with a variety of human diseases including multiple sclerosis, cardiovascular and neurological diseases. Human Cyclophilin D is a known MPTP regulator and although devoid of deep orthosteric sites, it is believed it can be targeted to develop clinically relevant candidates. Fragment-based drug discovery is a powerful approach for such drug-targets and we revisited a panel of 52 fragment hits by X-ray crystallography, which remained structurally elusive from a previous fragment screening campaign. Using two CypD mutants in a systematic parallel approach combining co-crystallization and soaking techniques (FragInc approach - from Fragment Incubation), new high-resolution crystal structures of CypD-fragment complexes were obtained. We identified a pyrazolo[1,5-a]pyrimidin-2(1H)-one fragment binding to a cleft in-between the known CypD S1' and S2 pockets. These structural insights guided the chemical synthesis of four new molecules to address simultaneously the novel pyrazolo binding pocket and the S2 site, and their crystal structures in complex with CypD were determined. This work opens new perspectives for structure-based drug design of novel tri-vector inhibitors targeting human CypD and proposes a parallel multi-technique methodology to address low-affinity fragments by X-ray crystallography.