Evidence suggests that sebum content is important in skin disorders such as acne. However, sebum levels change depending on the external environment, and quantifying skin sebum levels is challenging. Here, we propose an optimal method for quantifying the facial sebum level. Four hundred and sixty participants (160 males and 300 females) aged 20-40 were enrolled in this study. A Sebumeter SM 810 was used to measure the sebum level at five facial locations: the forehead, the chin, the left cheek, the right cheek, and the nose. The participants were divided into two groups; one group underwent a one-time measurement (n=390, male: female=120: 270), and the other underwent three consecutive measurements (n=70, male: female=40: 30). The casual sebum level (CSL) was measured in all patients after a 30-min acclimatization; subsequently, the sebum removal process was conducted, followed by a resting period of 1h to determine the sebum excretion rate (SER). Spearman's correlation analysis and the Wilcoxon signed-rank test were used to compare the sebum level consistency and differences between the groups. Although three consecutive measurements better reflected the sebum content, the one-time measurement also represented the relative sebum level. One hour after sebum removal, the sebum level recovered to 70%-90%; thus, this method was applicable for use in SER quantification. Of the five testing points, the sebum content was highest in the nose and lowest in the cheeks (both left and right). In addition, the cheeks were the most stable sites in terms of testing points, testing times, and CSL/SER values. A one-time measurement of the CSL could represent the SER 1h after the sebum removal. In our cohort, the sebum level of males with oily skin was decreased at age 32-35, and that of males with non-oily skin increased at 28-35. The opposite trend was observed in female participants. Sebum measurement methods were assessed, including testing times, indices (interval of time) and sites in a conditioned external environment. A one-time measurement of the CSL 1h after sebum removal was sufficient to determine the sebum level and SER, and the cheeks are recommended as the testing site. Sex and skin type differences were observed in sebum level changes with age.