Organosilane hybrid materials are of interest in the development of diagnostic devices and drug-delivery applications. Here we report a spectroscopic study involving the chemical and structural modification of thiol-functionalised organosilica particles with aminosilane to produce a bifunctional silica hybrid. The aminosilane was revealed to be distributed throughout the microsphere as opposed to being surface-localised as is commonly reported for modifications of pure silica. Spectroscopic methods including NMR, XPS, Ninhydrin and gravimetric measurements were employed to investigate the surface and internal elemental composition of the particles independently. A multiplexed model bioassay is presented to demonstrate the advantage of organosilane bifunctionality, enabling separate covalent attachment strategies for both homogeneous incorporation of fluorescent dyes and surface-specific biomolecule attachment. This study represents an advance in the understanding of organosilane chemistry resulting in versatile materials with a range of functionalities for covalent attachment.