Objectives/Goals: To determine the safety and feasibility of single-pulse transcranial magnetic stimulation (spTMS) for assessing corticospinal tract (CST) excitability and integrity in infants with perinatal brain injury, bridging foundational neuroscience to potential early diagnosis and clinical interventions during critical neuroplasticity periods. Methods/Study Population: Nineteen infants with perinatal brain injury underwent 1–3 spTMS sessions at three developmental time points: 3–6 months, 12 ± 1 month, and 18 ± 1 month. spTMS targeted the primary motor cortex to elicit motor-evoked potentials (MEPs), recorded via electromyography (EMG) from bilateral wrist flexor muscles. Safety monitoring included heart rate (HR), respiratory rate (RR), the Modified Behavioral Pain Scale (MBPS), and caregiver feedback. Feasibility was evaluated based on the ability to elicit MEPs, the number of trials that elicited MEPs, and procedure tolerability. Pre- and post-spTMS physiological and behavioral data were analyzed using linear mixed-effects models (LMEM) to account for repeated measures within subjects. Results/Anticipated Results: Thirty-five spTMS sessions were conducted in 19 infants (mean age 8.75 ± 5.12 months) with perinatal brain injury, delivering 1936 pulses with a median inter-pulse interval of 24.7 seconds. Analysis with LMEM found no significant changes in HR (mean difference  =  0.51 bpm, p  =  0.81) or RR (mean difference  =  0.69 breaths/min, p  =  0.66). MBPS scores showed a small statistically significant increase (mean difference  =  0.57, p  =  0.046), but overall remained low (mean score change from 1.94 to 2.51 on 0–10 scale). The median change score was 0, and 18/35 sessions showed no change in MBPS, indicating low discomfort with TMS. No adverse events were reported during or after the sessions. The feasibility of eliciting MEPs in this population was confirmed, with 235 MEPs identified in 17/19 participants. Discussion/Significance of Impact: Understanding neurodevelopment after injury is crucial for early diagnosis and targeted rehabilitation. Our study demonstrates that spTMS is a safe, feasible tool for assessing motor pathways in infants with early brain injury, highlighting its potential for clinical translation in neurodevelopmental disorders, and offering a pathway to improved care.