Amyloid fibrils of the protein α-synuclein (αS) have recently been identified as a biomarker for Parkinson’s disease (PD). To detect the presence of these amyloid fibrils, seed amplification assays (SAAs) have been developed. SAAs allow for the detection of αS amyloid fibrils in biomatrices such as cerebral spinal fluid and are promising for PD diagnosis by providing a dichotomous (yes/no) response. The additional quantification of the number of αS amyloid fibrils may enable clinicians to evaluate and follow the disease progression and severity. Developing quantitative SAAs has been shown to be challenging. Here, we report on a proof-of-principle study on the quantification of αS fibrils in fibril-spiked model solutions of increasing compositional complexity including blood serum. We show that parameters derived from standard SAAs can be used for fibril quantification in these solutions. However, interactions between the monomeric αS reactant that is used for amplification and biomatrix components such as human serum albumin have to be taken into account. We demonstrate that quantification of fibrils is possible even down to the single fibril level in a model sample consisting of fibril-spiked diluted blood serum.