Despite the availability of PARP inhibitors for cancer therapy, a biomarker to clearly stratify patients for selection of this treatment remains lacking. Here we describe a radiotracer-based method that addresses this issue, using the novel compound [¹²⁵I]KX1 as a PARP-1–selective radiotracer that can accurately measure PARP-1 expression in vitro and in vivo. The pharmacologic properties of the PARP radiotracer [¹²⁵I]KX1 was characterized in multiple cell lines where single-agent sensitivity was correlated with [¹²⁵I]KX1 binding to PARP-1. In vivo evaluation of [¹²⁵I]KX1 verified in vitro results, validating PARP radiotracers to define PARP-1 enzyme expression as an in vivo biomarker. Notably, PARP-1 expression as quantified by [¹²⁵I]KX1 correlated positively with the cytotoxic sensitivity of cell lines evaluated with PARP inhibitors. Overall, our results defined a novel technology with the potential to serve as a companion diagnostic to identify patients most likely to respond therapeutically to a PARP inhibitor. Cancer Res; 76(15); 4516–24. ©2016 AACR.