Paper and board consisting partially or fully of recycled fibers is the second most used food contact material (FCM) for food packaging applications. In spite of their wide use, there are currently major challenges on the safety assessment of recycled paper and board FCMs including the current absence of a harmonized EU regulatory framework governing their control and manufacture, the huge amount of food contact chemicals (FCCs) migrating from recycled paper and board FCMs into food and the concerns regarding their chemical safety in food packaging applications. Combined chemical analysis and in-vitro bioassays are considered as the most promising and efficient way to tackle the aforementioned challenges. The complex chemical nature of recycled paper and board FCMs has led to a wide variability in protocols and strategies for the safety assessment of such, including sample preparation methods. This variability leads to challenges regarding the comparability and evaluation of protocols and ultimately standardization. Sample preparation is a very important step in the framework of safety assessment as it highly impacts the efficiency and reliability of the subsequent analysis. There are various challenges and limitations that should be taken into account when structuring, evaluating and standardizing the sample preparation methods for the combined succeeding analysis, including the complexity of chemical mixture of the recycled paper and board FCMs, compatibility of the experimental parameters with in-vitro bioassays and the appropriate identity and concentration of FCCs that resemble the real migration occurring to food in contact with FCMs. Therefore, guiding principles and appropriate steps to tackle the aforementioned challenges and limitations are presented. Furthermore, developed migration/sample preparation protocols for the different food types packaged are included and the impact of critical experimental parameters is discussed.