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We demonstrate the trapping and analysis of individual proteins using a portable optical fiber tweezer setup with a double-nanohole in a gold film coating the fiber’s end and aligned with the fiber core. The instrument was used to trap and analyze cytochrome c, carbonic anhydrase, bovine serum albumin, and PR65 (wild-type and various point mutants). This approach was compared with a free-space optical tweezer setup that requires alignment of the laser focus to the aperture, whereas the fiber-based approach is both portable and alignment-free, which holds promise for applications in antibody discovery, small molecule drug discovery, protein interaction analysis and other applications using the standard well-plate format.