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In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 μg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Graziella Eliza Ronsein reports financial support was provided by State of Sao Paulo Research Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)