: In the present study, mixing behavior in chaotic advection flow through a twisted duct has been analyzed by three different methods: secondary flow structures, Lyapunov exponent and vorticity vector. Laminar fluid flow through a twisted duct with square and rectangular cross-sections under steady, sinusoidal and pulsation flows in Fluent software is considered. The twisted duct consists of three bends and each bend is ninety degrees curved duct. The angle between the curvature planes of two successive bends is also ninty degrees. To show the chaotic behavior of the twsited duct flow, the secondary flow structures and Lyapunov exponent are studied. Based on these two criteria, the appearance of homoclinic and heteroclinic connections in the secondary flow structures and (or) the positive value of Lyapunov exponent show that the flow is chaotic. The more chaos in the flow represents mixing enhancement. Based on the secondary flow strucutures as a mixing criterion, elongation and displacement of elliptic orbits, the more variations of the secondary flow structures in the flow cycle and appearance of the homoclinic and heteroclinic connections show mixing improvement, but the numerical value of the mixing is not provided from this method. Therefore, two another methods, Lyapunov exponent and vorticity vector, are analyzed to calculate the value of mixing. Based on the method of Lyapunov exponent which shows the stretching and folding in the flow, the positive and more value of the parameter shows the mixing improvement. Based on the method of vorticity vector, which shows stretching and engulfment in the flow, the increasing of the three vector components shows mixing enhancement. The results of the three methods were the same and showed that homoclinic connections appears and becomes prominent in the secondary flow structures under the steady flow and this shows the chaos existence and mixing improvement in the flow. Also, the results presented that the mixing obtained in sinusoidal flow is lower than the steady flow. But, by combination of the steady and sinusoidal flows, mixing improves. In the pulsation flow, mixing increases with velocity component ratio(?), and the lower values of Womersley number(?) provides better mixing. Mixing increases with Reynolds number in the steady flow, whereas in the pulsation flow it has not a deterministic behavior with Reynolds number. The pulsation flow enhances the mixing over the steady flow when ??2. Also, mixing has an oscillation behavior along the duct length which is due to the coexistence of the regular and chaotic zones in the flow. In addition to the twisted duct, a chaotic micromixer has been also designed and its operating range in the steady and pulsation flows has been analyzed. Mixing behavior of the flow in the micromixer has been studied based on the three criteria: concentration distribution, Lyapunov exponent and vorticity vector. The results showed that chaotic advection enhances the mixing without significant increasing of the pressure drop, and full mixing is achieved in the steady flow when Re=50. Also, the pulsation flow enhances the mixing over the steady flow when ??2, and mixing decreases with Strouhal number and its reduction rate decreases with Strouhal number, too. Keywords: Mixing, Chaotic advection, Secondary flow structure, Lyapunov exponent, Vorticity vector, Twisted duct, Micromixer.