Microfluidic valve systems show great potential to automate mixing, dilution, and time-resolved reagent supply within biochemical assays and novel on-chip cell culture systems. However, most of these systems require a complex and cost-intensive fabrication in clean room facilities, and the valve control element itself also requires vacuum or pressure sources (including external valves, tubing, ports and pneumatic control channels). Addressing these bottlenecks, the herein presented biocompatible and heat steam sterilizable microfluidic valve system was fabricated via high-resolution 3D printing in a one-step process – including inlets, micromixer, microvalves, and outlets. The 3D-printed valve membrane is deflected via miniature on-chip servomotors that are controlled using a Raspberry Pi and a customized Python script (resulting in a device that is comparatively low-cost, portable, and fully automated). While a high mixing accuracy and long-term robustness is established, as described herein the system is further applied in a proof-of-concept assay for automated IC₅₀ determination of camptothecin with mouse fibroblasts (L929) monitored by a live-cell-imaging system. Measurements of cell growth and IC₅₀ values revealed no difference in performance between the microfluidic valve system and traditional pipetting. This novel design and the accompanying automatization scripts provide the scientific community with direct access to customizable full-time reagent control of 2D cell culture, or even novel organ-on-a-chip systems.