The light spectrum above 650 nm allows for good tissue penetration depths, therefore far-red and near-infrared fluorescent dyes are popular fluorophores applied in (bio)medical diagnostics, including image-guided surgery. Unfortunately, near-infrared fluorescent dyes often suffer from instability and limited brightness, two important features that, together with the labelling efficiency (e.g., non- one- or di-conjugated products) and serum-dye interactions are key elements that drive in vivo characteristics. Due to the fact that stability and brightness of far-red fluorophores are often superior over near-infrared dyes, interest in the use of dyes such as Cy5 is increasing. As there are clear indications that the chemical structure of a dye influences the (photo)physical properties, these properties of ten structural variants of asymmetrical Cy5-(R1)R2-(R3)COOH (R representing the varied substituents) dyes were extensively studied. While stacking in solution was not induced in most of the Cy5 far-red dyes, multimers and stacking characteristics were observed in protein conjugates. Although all dye variants were shown to be stable towards photobleaching, clear differences in brightness and serum interactions were found. Combined, these findings indicate that there is a direct relation between chemical substituents and the properties of Cy5 dyes, and that this feature should be considered when using fluorescent dyes in future tracer development. Display omitted •Clear differences in brightness were found upon changing the fluorophore substituents.•The investigated fluorophores were optical and chemically stable.•Increasing number of sulfonates on the Cy5 decreased non-specific serum protein interactions.•Clear differences in labelling efficiencies between the dyes were found during protein conjugation.