IMPORTANCE Binocular indirect ophthalmoscopy (BIO) examination for retinopathy of prematurity (ROP) is a well-known cause of repeated preterm infant stress. OBJECTIVE To compare stress during investigational optical coherence tomography (OCT) imaging to that during BIO for ROP. DESIGN, SETTING, AND PARTICIPANTS This cross-sectional study examined infants at the bedside in the intensive care nursery. Consecutive preterm infants enrolled in Study of Eye Imaging in Preterm Infants (BabySTEPS) who had any research OCT imaging as part of the study. Patients were recruited from June to November 2019, and analysis began April 2020. MAIN OUTCOMES AND MEASURES Infant stress was measured using modified components of a neonatal pain assessment tool before (baseline) and during OCT imaging and BIO examination of each eye. RESULTS For 71 eye examinations of 16 infants (mean [SD] gestational age, 27  weeks; birth weight, 869  g), change from baseline to each eye examination was lower during OCT imaging than during BIO and the difference between OCT imaging and BIO at each eye examination was significant for the following: infant cry score (first eye examination: mean [SD], 0.03 [0.3] vs 1.68 [1.2]; -1.65 [95% CI, -1.91 to -1.39]; second eye examination: mean [SD], 0.1 [0.3] vs 1.97 [1.2]; -1.87 [95% CI, -2.19 to -1.54]), facial expression (first eye: 3 [4%] vs 59 [83%]; -79% [95% CI, -87% to -72%]; second eye: 4 [6%] vs 61 [88%]; -83% [95% CI, -89% to -76%]), and heart rate (first eye: mean [SD], -7  vs 13 ; -20 [95% CI, -26 to -14]); second eye: mean [SD], -3  vs 20  beats per minute; -23 [95% CI, -29 to -18]) (P <.001 for all). Change in respiratory rate and oxygen saturation did not differ between OCT imaging and BIO. CONCLUSIONS AND RELEVANCE While the role of OCT alone or in combination with BIO is currently unknown for ROP, these findings suggest that investigational OCT imaging of ROP is less stressful than BIO examination by a trained ophthalmologist.