An identified impediment to the advancement of science in the field of proteomics is the deterioration of proteins in tissue upon removal of the tissue from its natural state. To reduce this degradation, human tissues are frozen and stored in either liquid nitrogen or -80°C environments. It is believed that frozen tissue in ultralow temperatures preserves proteins against enzyme degradation. Various molecular, biophysical, and biochemical analytical studies require that frozen tissues be thawed before being used for analyses. Depending on downstream analyses, tissues are thawed at different temperatures (37°C, room temperature or 4°C). However, there is very little literature that describes the effects of different thaw temperatures on enzymatic inactivation in tissue lysates. We investigated the effects of preprocessing variable thaw temperature on postprocessed lysates using tyrosine phosphatase and phosphatase and tensin homolog activity assays. In our study we examined the thawing of frozen human thyroid tissues at the traditional temperatures of 4°C (on ice), 37°C (in an oven), and the novel temperature of 95°C (using Stabilizor T1™). The tissue lysates were processed without the addition of enzymatic inhibitors. Our results showed that in benign, malignant, and diseased tissues, high temperature thawing is effective in reducing enzymatic activity. In normal tissue, the reduction is dependent on individual enzymes. This suggests that if tissue lysates are to be obtained from frozen tissues without the addition of inhibitors, high temperature thawing might have marked improvement in downstream non-enzymatic analyses of diseased and neoplastic tissues.