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To compare vitreous biopsy methods using analysis platforms used in proteomics biomarker discovery.Vitreous biopsies from 10 eyes were collected sequentially using a 23-gauge needle and a 23-gauge vitreous cutter instrument. Paired specimens were evaluated by UV absorbance spectroscopy, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS).The total protein concentration obtained with a needle and vitrectomy instrument biopsy averaged 1.10 mg/mL (standard error of the mean = 0.35) and 1.13 mg/mL (standard error of the mean = 0.25), respectively. In eight eyes with low or medium viscidity, there was a very high correlation (R2 = 0.934) between the biopsy methods. When data from 2 eyes with high viscidity vitreous were included, the correlation was reduced (R2 = 0.704). The molecular weight protein sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles of paired needle and vitreous cutter samples were similar, except for a minority of pairs with single band intensity variance. Using LC-MS/MS, equivalent peptides were identified with similar frequencies (R2 ≥ 0.90) in paired samples.Proteins and peptides collected from vitreous needle biopsies are nearly equivalent to those obtained from a vitreous cutter instrument. This study suggests both techniques may be used for most proteomic and biomarker discovery studies of vitreoretinal diseases, although a minority of proteins and peptides may differ in concentration.