<p> Collagen is the main component of human connective tissue and extracellular matrix. Here we report multiple novel methods for utilizing optical tweezers to measure mechanical properties of different hierarchical levels of collagenous materials. First, we introduce a method for optical trap calibration that is suitable for viscoelastic material. The method is designed for use on experimental setups with two optical tweezers and is based on pulling a trapped particle with one trap while holding it with the other. The method combines advantages of commonly known PSD-fitting and fast-sweeping methods, allowing calibration of a completely fixed trap in a fluid of unknown viscosity/viscoelasticity without additional expensive equipment. Then we report an approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found <i>in vivo</i> and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. The approach also avoids the traditional drying-soaking cycle, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. Lastly, we report a new method for characterizing anisotropic viscoelastic response of collagenous matrices. Anisotropic collagenous extracellular matrices are used in biomedicine to enhance the wound healing process by directing fibroblast proliferation. We utilize an optical trap to monitor the thermal fluctuations of microspheres embedded into collagenous network to extract a viscoelastic response function of the network along the principal axes of anisotropy.</p>