Little is known about the pre-analytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles as tissue reacted to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability, and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (±20% from the time zero sample) of signal pathway phosphoprotein levels, as well as decreases over time independent of tissue type, post-translational modification, and protein sub-cellular location (tissues: breast, colon, lung, ovary, uterus (endometrium/myometrium, metastatic melanoma)). Comparison across tissue specimens showed >20% decrease of AKT S473 (p<0.002) and MARCKS S152/156 (p<0.0001) within the first 90 minutes post excision. Proteins in apoptotic (Cleaved Caspase 3 D175(p<0.001)), proliferation/survival/hypoxia (IRS-1 S612(p<0.0003), AMPKß S108(p<0.005), ERK T202/Y204(p<0.003), GSK3aß S21/9(p<0.01)), and transcription factor pathways (STAT1 Y701(p<0.005), CREB S133(p<0.01)) showed >20% increases within ninety minutes post procurement. eNOS S1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 T71, SAPK/JNK T183/Y185, STAT1 Y701, JAK1 Y1022/1023, and Pak1/Pak 2 S199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.