Reference techniques to study rumen biohydrogenation (BH) rely on the comparison of intake and duodenal or (ab)omasal flows of polyunsaturated fatty acids (PUFA), whereas the net BH of PUFA to their saturated end-products gives a quantitative measure of accumulating BH intermediates. The current review paper aims at evaluating alternative in vivo, in vitro and in sacco techniques to simulate reference in vivo results of unprotected PUFA sources, as well as strategies for overcoming or manipulating BH. In vivo rumen sampling approaches show potential but require further investigation, whereas in sacco results are inappropriate. In vitro 24-h batch incubations and continuous cultures approach in vivo BH of unprotected C-18 PUFA sources and are useful to assess the pH value at which dissociation of calcium salts of fatty acids occurs, but overestimate the degree of rumen inertness of formaldehyde-treated oil(seeds) and marine products. Batch or continuous cultures provide an accurate estimate (0.6) of the proportion of hydrogenated C-18 PUFA that are converted into their saturated end-product, except for incubations with high levels of fermentable substrate (> 1.0 g/100 mL) in combination with high amounts of C-18 PUFA(> 0.5 mg/mL) or in incubations with EPA and DHA.