Drug toxicity is a long-standing concern of modern medicine. A typical anti-pain/fever drug paracetamol often causes hepatotoxicity due to peroxynitrite ONOO−. Conventional blood tests fail to offer real-time unambiguous visualization of such hepatotoxicity in vivo. Here we report a luminescent approach to evaluate acute hepatotoxicity in vivo by chromophore-conjugated upconversion nanoparticles. Upon injection, these nanoprobes mainly accumulate in the liver and the luminescence of nanoparticles remains suppressed owing to energy transfer to the chromophore. ONOO− can readily bleach the chromophore and thus recover the luminescence, the presence of ONOO− in the liver leads to fast restoring of the near-infrared emission. Taking advantages of the high tissue-penetration capability of near-infrared excitation/emission, these nanoprobes achieve real-time monitoring of hepatotoxicity in living animals, thereby providing a convenient screening strategy for assessing hepatotoxicity of synthetic drugs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim