In　vivo　levels　of　reactive　oxygen　species　(ROS)　and　reactive　nitrogen　species　(RNS)　are　critical　to　many　physiological　and　pathological　processes.　Because　of　the　distinct　differences　in　their　biological　generation　and　effects,　simultaneously　visualizing　both　of　them　could　help　deepen　our　insights　into　the　mechanistic　details　of　these　processes.　However,　real-time　and　deep-tissue　imaging　and　differentiation　of　ROS-　and　RNS-related　molecular　events　in　living　subjects　still　remain　a　challenge.　Here,　we　report　the　development　of　two　activatable　19F　magnetic　resonance　imaging　(MRI)　molecular　probes　with　different　19F　chemical　shifts　and　specific　responsive　behaviors　for　simultaneous　in　vivo　detection　and　deep-tissue　imaging　of　O2•-　and　ONOO-.　These　probes　are　capable　of　real-time　visualization　and　differentiation　of　O2•-　and　ONOO-　in　living　mice　with　drug-induced　acute　kidney　injury　by　interference-free　multiplexed　hot-spot　19F　MRI,　illustrating　the　potential　of　this　technique　for　background-free　real-time　imaging　of　diverse　biological　processes,　accurate　diagnosis　of　various　diseases　in　deep　tissues,　and　rapid　toxicity　evaluation　of　assorted　drugs.　©　2021　American　Chemical　Society.