In contrast to conventional functional magnetic resonance imaging (fMRI) analysis across participants, item analysis allows generalizing the observed neural response patterns from a specific stimulus set to the entire population of stimuli. In the present study, we perform an item analysis on an fMRI paradigm (EmpaToM) that measures the neural correlates of empathy and Theory of Mind (ToM). The task includes a large stimulus set (240 emotional vs. neutral videos to probe empathic responding and 240 ToM or factual reasoning questions to probe ToM), which we tested in two large participant samples (N = 178, N = 130). Both, the empathy-related network comprising anterior insula, anterior cingulate/dorsomedial prefrontal cortex, inferior frontal gyrus, and dorsal temporoparietal junction/supramarginal gyrus (TPJ) and the ToM related network including ventral TPJ, superior temporal gyrus, temporal poles, and anterior and posterior midline regions, were observed across participants and items. Regression analyses confirmed that these activations are predicted by the empathy or ToM condition of the stimuli, but not by low-level features such as video length, number of words, syllables or syntactic complexity. The item analysis also allowed for the selection of the most effective items to create optimized stimulus sets that provide the most stable and reproducible results. Finally, reproducibility was shown in the replication of all analyses in the second participant sample. The data demonstrate (a) the generalizability of empathy and ToM related neural activity and (b) the reproducibility of the EmpaToM task and its applicability in intervention and clinical imaging studies. © 2020 The Authors. Human Brain Mapping published by Wiley Periodicals, Inc.