Sequential phosphorus (P) extraction (SPE) is a well-established and widely applied method for quantitatively and qualitatively determining the critical nutrient P in freshwater sediments. It allows the estimation of P bioavailability and facilitates the evaluation of the long-term effects of eutrophication mitigation measures. Most current protocols do not differentiate between redox-sensitive Fe(III)-P and more stable reduced Fe(II)-P minerals, such as vivianite. In this study, we tested a modified SPE protocol designed to quantify Fe(II)-P (vivianite-P) as a separate phase through the complexation of Fe(II) with 2,2′-bipyridine (Bipy). Seven lakes were selected as study sites with different sedimentary Fe and P contents and restoration histories. We validated the Bipy extraction step through direct comparison with results from the conventional protocol and the application of direct mineral detection methods, including x-ray absorption near-edge structure at the Fe and P K-edges, x-ray diffraction, optical microscopy, and scanning electron microscopy with energy dispersive x-ray spectroscopy. The Bipy fraction was primarily extracting P that was conventionally extracted in the bicarbonate-dithionite (redox-sensitive Fe/Mn-bound) and NaOH (metal-[Fe/Al-]bound) fractions. The results from the direct detection methods indicated that the extracted Fe(II)-P was predominantly vivianite. The efficiency of the Bipy extraction was decreased in samples with high crystallinity, but excessive Fe(II) or high organic content had minimal impact. Hence, it is highly recommended to use x-ray diffraction and x-ray absorption near edge structure in combination with the modified extraction protocol. Overall, the method tested with different freshwater sediments provides robust results when quantification of Fe(II)-P including vivianite is an important objective.