The conception of inverse methods in the context of Traction Force Microscopy (TFM) is influenced by multiple factors, such as nonlinear effects, dimensionality (2D/3D), and regularization/constraints, amongst others. Solving the inverse problem often requires the inversion of a matrix, and the presence of noise in the measured displacements can lead to unrealistic reconstructed tractions. To address this issue, Tikhonov regularization is commonly used, penalizing high norm values of computed tractions. The aim of this study is to compare the performance of different inverse methodologies (including some original variations) in 3D TFM, considering constraint imposition and regularization along the formulation. The different methodologies are numerically elaborated within a novel combined Newton–Raphson/Finite Element Method scheme that provides converged solutions in few iterations. The impact of constraint imposition and regularization in traction reconstruction is evaluated in terms of accuracy, efficiency (CPU time), and inherent characteristics of the methodology. Results show that, applying regularization and constraints (based on the fulfillment of fundamental principles) provides the best traction reconstruction, while simultaneously ensuring an optimum estimate of the maximum traction, at the cost of high CPU time and low efficiency. Moreover, regularization-based methods introduce the challenge of calibrating the regularization parameter, usually done under subjective criteria. On the other hand, non-regularized but constrained methods may represent a nice compromise between accuracy and efficiency, while avoiding pre-processing and calibration of such regularization parameter. It is emphasized the importance of considering not only traction reconstruction quality but also the efficiency and complexity of implementation (intrusiveness) when selecting an appropriate inverse method for TFM analysis.