Purpose: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemistry (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. Experimental Design: PTEN IHC was validated by employing formalin fixed and paraffin-embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution single nucleotide polymorphism microarray analysis was done on a subset of these cases. Results: PTEN protein loss, as assessed as a dichotomous IHC variable, was highly reproducible, correlated strongly with adverse pathologic features (e.g., Gleason score and pathologic stage), detected between 75% and 86% of cases with PTEN genomic loss, and was found at times in the absence of apparent genomic loss. In a cohort of 217 high risk surgically treated patients, PTEN protein loss was associated with decreased time to metastasis. Conclusion: These studies validate a simple method to interrogate PTEN status in clinical specimens and support the utility of this test in future multicenter studies, clinical trials, and ultimately perhaps for routine clinical care.