Isotretinoin is a widely prescribed drug for various dermatological conditions, especially acne. Blood tests are routinely performed to monitor its side effects. However, the optimal testing schedule remains controversial. To evaluate the laboratory monitoring tests and schedules of patients with acne taking isotretinoin to determine the most efficient testing regimen. We retrospectively reviewed electronic medical records from the Dermatology Department of Dongguk University Ilsan Hospital from 2005 to 2020 for patients prescribed isotretinoin for acne who underwent monthly blood tests. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglyceride (TG) levels significantly changed between 5 and 6 mo when the total prescription period and dose variables were considered altogether. The high-density lipoprotein level also significantly changed between 5 and 6 mo. However, low-density lipoprotein (LDL) and total cholesterol levels significantly changed between 1 and 2 mo. We recommend testing AST, ALT, and TG levels once every 5 to 6 mo. We also suggest testing LDL and total cholesterol levels during the first and second months.