Background: Cytostatic drugs are increasingly being prepared with a cytostatic robot, though it is not known whether the dose of the final product is more accurate after automated or manual preparation. This study is the first to compare accuracy and precision of automated preparations with manual preparations by measuring volumes and drug concentrations. Methods: The accuracy and precision of automated and manual preparations were compared by gravimetric and concentration measurements. During ten days 80 solutions were prepared; 40 robot preparations and 40 manual preparations. With both preparation methods, 20 methotrexate (MTX) and 20 cyclophosphamide (CP) bags were compounded. We simulated normal working conditions by performing the preparations on Monday till Friday. The MTX and CP concentrations were measured with validated ultra high performance liquid chromatography (UHPLC) methods on the last preparation day. Results: With UHPLC analysis, dose accuracy (mean dose error) of robotic or manual preparation of MTX were 1.70% and 0,96% respectively. With gravimetric analysis, these values were 0.50% and 1.96%. Precision (standard error) of the robotic preparation for MTX was significantly smaller than that of manual preparation (p < 0.001). Dose accuracy (mean dose error) of robotic or manual preparation of CP, with UHPLC analysis, were 6.10% and 5.20% respectively. With gravimetric analysis, these values were 0,67% and 0,18%. Conclusion: We conclude that both robotic and manual compounding produce accurate cytostatic products in which the mean percentage of active substance differs by less than 10% from the prescribed amount. Both preparation methods are compliant with the Dutch Medicines Act and the European Pharmacopoeia.