In unconscious patients, pupillary light reflex is an indicator of brain damage. In the current study, a smartphone application was developed for the purpose of measuring pupillary light reflex with an aim to determine the agreement between pupillary light reflex measurements using a smartphone application (APP) and a penlight (PEN). The APP acquires five sequential photographs using the camera flash in order to stimulate the pupil. The initial image is captured prior to the flash, and the subsequent image is obtained while the flash is on. The remaining three images are captured whilst the flash is off. Pupillary right reflex was assessed in 30 healthy subjects using a PEN. After 10 min, the examiners inspected the images of light reflex acquired from the same subjects using the APP, and completed the corresponding questionnaire containing details of pupil size and degree of response. Agreement between the two assessment methods was determined by calculating bias, limits of agreement, and the intraclass correlation (ICC) coefficient. A statistically significant difference was not observed between the two methods regarding pupil size and degree of response. Bias was 0.1 mm and limits of agreement were ±1.5 mm, as compared with PEN. ICC was 0.93 (95% confidence interval, 0.89-0.96). Therefore, it may be concluded that the results of pupillary light reflex assessed by PEN and APP display no significant difference. Furthermore, the APP provides advantages such as portability, objectivity and the possibility of being used as objective medical evidence.