Microfluidic technology can provide cost-efficient, user-friendly devices for applications in the life sciences, environmental monitoring and the agrifood, through miniaturization and automation. In Alberta, undetected estrus is the leading reason for poor reproductive efficiency in dairy herds. Therefore, accurately identifying estrus by monitoring related hormones will be ultimately beneficial. Herein, we developed an automated centrifugal microfluidic disc aimed at realizing low-cost, easy-to-use, rapid on-site hormone detection in aqueous solutions. The workflow is designed with direct delivery of milk to the device, integrated sample cleanup, followed by ondisc analysis with embedded color sensor films and a simple camera.