Preanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally, two different protein extraction protocols, using different extraction buffers (8 M urea buffer, or Bio-Rad buffer without urea) were tested. For snap-frozen samples, protein extraction yields were comparable with the two buffer systems. For heat-stabilized samples, total protein yields were significantly lower following extraction in non-urea buffer. However, the concentrations of specific phosphoproteins were significantly higher in heat-stabilized samples than in the corresponding snap-frozen samples, indicating that this tissue processing method better preserved phosphoproteins. Significant differences were found between the measured phosphoprotein levels in heat-stabilized and snap-frozen tissue, suggesting that alterations occur very rapidly after tissue excision. Our results suggest that heat stabilization can be used as a tissue processing method for subsequent phosphoprotein analyses, but also suggest that the BioPlex phosphoprotein assay could be used as a possible quality control method to assess tissue sample integrity.