Activating mutations of Src homology-2 domain-containing protein tyrosine phosphatase-2 (Shp2) cause multiple childhood conditions for which there is an unmet therapeutic need, including juvenile myelomonocytic leukemia (JMML) and Noonan syndrome. Using unbiased proteomics, the protein targets of SFX-01, an α-cyclodextrin-stabilized isothiocyanate complex that covalently adducts cysteines that is in clinical development, were identified and included Shp2. SFX-01 induced an inhibitory dithiolethione modification at the Shp2 active site cysteine. Importantly, in a transgenic mouse model of human Noonan syndrome with hyperactive D61G Shp2, SFX-01 concomitantly normalised their phosphatase activity and myeloid cell count. Furthermore, SFX-01 also attenuated JMML human patient-derived hematopoietic stem cell proliferation that was linked to STAT1 signaling and decreased cyclin D1 expression, resulting in cell-cycle arrest. We conclude that SFX-01 is an activating mutant Shp2 inhibitor and may offer beneficial effects in patients with JMML or Noonan syndrome.