Characterising the asphaltene and carboxylic acid (naphthenic acid) content of crude oil is important for petroleum production, transport, storage and environmental science. This is because, the proportion of asphaltene and the concentration of acidic compounds in petroleum can be used to characterise viscosity (e.g. producibility), refining potential (e.g. its value) and chemical recalcitrance and thus behaviour as a contaminant. Here we present an assay for determining the proportion of asphaltene and total acid number (TAN) of petroleum. The method utilises a microfluidic component called an H-cell and produces an asphaltene-free fraction, either hydrocarbon or methanol-soluble, that can be forwarded for further advanced analysis and used to determine asphaltene content and TAN value. The H-cell method depends on a diffusion-based separation that is only practical when a sample is manipulated at a microscale and thus is fundamentally different to previous methods for assaying these parameters that utilise solubility- or chromatography-based methods. Comparisons of asphaltene and TAN measurements derived from the H-cell based assay have very high correlations with the ASTM D4124 and ASTM D974-97 methods. Therefore rapid and simultaneous determination of asphaltene content and TAN value can be achieved by an H-cell based format. While this format is suited to miniaturisation and point of need analysis, the main benefit of the H-cell method might be its capacity to provide new analytical windows.