Irrational use of drugs remains a major challenge especially in developing countries, which contributed to a heavy pharmaceutical expenditure burden. Price regulation has been taken to curb the growth of pharmaceutical expenditures in many countries. This study aimed to investigate the impact of different mark-up drug policies on drug-related expenditures in tertiary public hospitals in Shanghai, China. Data were drawn from the audited financial statement in 24 tertiary public hospitals in Shanghai from January 2015 to December 2018. Drug-related revenue data and per capita cost data pre- and post-intervention were included. Interrupted time series design was applied to assess the actual effects of Fixed Percent Mark-up Drug (FPM) policy and Zero Mark-up Drug (ZMD) policy respectively. Results showed that ZMD policy achieved better intervention effects on declining drug-related expenditures than FPM policy. Apart from a declining trend in drug proportion (coefficient = -0.0017, p = 0.031), no other significant changes were found during FPM implementation. However, ZMD policy was associated with a level decline in per capita outpatient drug cost (coefficient = -12.21, p = 0.025) and a trend decline in per capita inpatient drug cost (coefficient = -25.12, p < 0.001), as well as a level decrease (coefficient = -0.0256, p = 0.001) and a downward tendency (coefficient = -0.0018, p < 0.001) in drug proportion. ZMD policy was effective in regulating drug-related expenditures, while FPM policy was difficult to achieve expected results due to the existence of profit space. Further regulation should be strengthened in the future, especially on drug revenue and per capita drug cost.