Critical energy release rate for delamination initiation in composites as a material property, supposed to be independent from non-material variables. However, a thorough literature review presented in this study shows that in many cases it may vary with the variation of layup configuration or geometrical and dimensions. This study is aimed to investigate the effect of interface layers orientation on fracture toughness by eliminating the other influential parameters such as stacking sequence, by selecting the anti-symmetric layup configuration of Double Cantilever Beam, [±/010/±]as, in which will be 0°, 30°, 45° and 60°. The energy release rates data have been calculated using different criteria and techniques to obtain the load and displacement at initial crack growth and the results were compared with the standard methods. The damage zone near the crack tip is also illustrated before and after the crack propagation by microscopic images of delamination front, and discussed for all investigated interface fiber angles. Experimental results show that the effect of interface layers orientation on fracture toughness of the investigated layup configurations based on the nonlinear technique as a standard procedure is negligible while other techniques show a considerable changes in the calculated energy release rate with the increase of interface layers angle from zero to 60 degrees.