Cell　survival　rate　determines　engraftment　efficiency　in　adipose-derived　mesenchymal　stem　cell　(ADSC)-based　regenerative　medicine.　In　vivo　monitoring　of　ADSC　viability　to　achieve　effective　tissue　regeneration　is　a　major　challenge　for　ADSC　therapy.　Here,　we　developed　an　activated　near-infrared　II　(NIR-II)　fluorescent　nanoparticle　consisting　of　lanthanide-based　down-conversion　nanoparticles　(DCNPs)　and　IR786s　(DCNP@IR786s)　for　cell　labeling　and　real-time　tracking　of　ADSC　viability　in　vivo.　In　dying　ADSCs　due　to　excessive　ROS　generation,　absorption　competition-induced　emission　of　IR786s　was　destroyed,　which　could　turn　on　the　NIR-II　fluorescent　intensity　of　DCNPs　at　1550　nm　by　808　nm　laser　excitation.　In　contrast,　the　NIR-II　fluorescent　intensity　of　DCNPs　was　stable　at　1550　nm　by　980　nm　laser　excitation.　This　ratiometric　fluorescent　signal　was　precise　and　sensitive　for　tracking　ADSC　viability　in　vivo.　Significantly,　the　nanoparticle　could　be　applied　to　quantitively　evaluate　stem　cell　viability　in　real-time　in　vivo.　Using　this　method,　we　successfully　sought　two　small　molecules　including　glutathione　and　dexamethasone　that　could　improve　stem　cell　engraftment　efficiency　and　enhance　ADSC　therapy　in　a　liver　fibrotic　mouse　model.　Therefore,　we　provide　a　potential　strategy　for　real-time　in　vivo　quantitative　tracking　of　stem　cell　viability　in　ADSC　therapy.　©　2022　American　Chemical　Society