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Although automated methods for stroke lesion segmentation exist, many researchers still rely on manual segmentation as the gold standard. Our detailed, standardized protocol for stroke lesion tracing on high-resolution 3D T1-weighted (T1w) magnetic resonance imaging (MRI) has been used to trace over 1,300 stroke MRI. In the current study, we describe the protocol, including a step-by-step method utilized for training multiple individuals to trace lesions (“tracers”) in a consistent manner and suggestions for distinguishing between lesioned and non-lesioned areas in stroke brains. Inter-rater and intra-rater reliability were calculated across six tracers trained using our protocol, resulting in an average intraclass correlation of 0.98 and 0.99, respectively, as well as a Dice similarity coefficient of 0.727 and 0.839, respectively. This protocol provides a standardized guideline for researchers performing manual lesion segmentation in stroke T1-weighted MRI, with detailed methods to promote reproducibility in stroke research.