Updated project metadata. Formalin-fixed paraffin-embedded tissues are suitable for proteomic and phosphoproteomic biomarker studies by data-independent acquisition mass spectrometry. The sample preparation method chosen influences the number, intensity, and reproducibility of identifications. By comparing the performance of four deparaffinisation and rehydration methods, we found that heptane and methanol quantified the most protein groups whilst the most widely used xylene and ethanol quantified the least. We then modified five extraction methods from the literature based on their diverse lysis and digestion conditions before evaluating their performance using rat organs of the kidney, leg muscle, lung, and testis. All methods performed well except for SP3, that struggled with tissue lysis. Heat n’ Beat was the fastest, had the highest digestion efficiency, quantified protein groups, and lowest coefficient of variation across replicates. Strap produced the highest peptide yield and SPEED produced a ultraviolet quantitation interference. TFE produced the highest phosphoprecursor intensity with a 10-15% higher enrichment efficiency. We demonstrate that the choice of extraction method has a larger impact on the phosphoproteome than the proteome. We conclude by providing recommendations for choosing the most appropriate method for different settings.