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Accurate assessment of fiber-matrix interface shear strength may play key role in understanding structural strength behavior of polymer-matrix composites, and provide additional flexibility for enabling high-performing material designs. This work presents a method for assessment of fiber-matrix interface strength in carbon fiber reinforced polymers (CFRPs). The method uses in-situ scanning electron microscopy (SEM) imaging while pushing individual fibers out of 20–30 μm thick membranes. Femtosecond laser machining has been utilized to address standing challenges associated with manufacturing high-quality fiber push-out specimens. To ensure that the specimen preparation method used did not affect the properties of the composite, a thorough study has been conducted using benchmark specimens. The benchmark specimens are prepared using a conventional lapping/polishing process. Results for two CFRPs compare fiber-matrix interface shear strength generated using free-standing samples (conventional lapping/polishing preparation) and femtosecond laser-machined specimens. Both specimen types exhibit similar strength. However, load-displacement response of the free-standing samples is overly compliant, representing specimen response rather than fiber-matrix interface material behavior. The excessive compliance is caused by inherent imperfections in the contact of the sample and the test fixture. The femtosecond laser-machined samples are free of such imperfections. Finite element analysis has been accomplished to better understand the load-displacement response.