AIM: To characterize the phenylephrine test in aponeurotic and congenital eyelid ptosis, to determine the appropriate timing of the phenylephrine test, and to assess the responses of the upper and lower eyelids. METHODS: This was a retrospective analysis of 140 eyes of 87 patients (mean age 52.29±16.45y; 22 males, 65 females) with upper eyelid ptosis. Totally 88.6% had aponeurotic and 11.4% had congenital ptosis. For the evaluation of the responses of the upper and lower eyelids to topical 2.5% phenylephrine, the scleral show height, the marginal reflex distance (MRD) between the inferior margin of the upper eyelid and pupillary light reflex (MRD1), and between the central portion of the lower eyelid and pupillary light reflex (MRD2) were measured at the 2nd, 5th, and 15th minutes. The changes of MRD1 and MRD2 with time (ΔMRD1 and ΔMRD2) were evaluated. RESULTS: The mean MRD1, MRD2, and scleral show heights increased within 5min after testing, remaining largely stable between the 5th-15th minutes. The percentage of eyes with a greater response in MRD1 increased with increased severity of ptosis (P<0.05). Eyes with aponeurotic ptosis were more responsive to phenylephrine testing than congenital ptosis. The mild ptosis group had lower scleral show measurements and higher ΔMRD2 values. The ΔMRD1 and ΔMRD2 values were poorly correlated in all measurement times. CONCLUSION: Performing the phenylephrine test 5min after instilling the reagent is adequate to assess the maximum response of the upper and lower eyelids. The upper and lower eyelid responses in phenylephrine testing are poorly correlated. However, the ΔMRD2 is related with baseline scleral show degree that may be a postoperative predictive factor. Further studies are necessary to determine the relationship between the responses of the lower eyelids to phenylephrine testing.