Single-file focusing and minimum interdistance of micron-size objects in a sample is a prerequisite for accurate flow cytometry measurements. Here, we report analytical models for predicting the focused width of a sample stream b as a function of channel aspect ratio α, sheath-to-sample flow rate ratio f and viscosity ratio λ in both 2D and 3D focusing. We present another analytical model to predict spacing between an adjacent pair of objects in a focused sample stream as a function of sample concentration C, mobility ϕ of the objects in the prefocused and postfocused regions and flow rate ratio f in both 2D and 3D flow focusing. Numerical simulations are performed using Ansys Fluent VOF model to predict the width of sample stream in 2D and 3D hydrodynamic focusing for different sample-to-sheath viscosity ratios, aspect ratios and flow rate ratios. Experiments are performed on both planar and three-dimensional devices fabricated in PDMS to demonstrate focusing of sample stream and spacing of polystyrene beads in the unfocused and focused stream at different sample concentrations C. The predictions of the analytical model and simulations are compared with experimental data, and a good match is found (within 12 %). Further, mobility of objects is experimentally studied in 2D and 3D focusing, and the spread of the mobility data is used as tool for the demonstration of particle focusing in flow cytometer applications. © 2016, Springer-Verlag Berlin Heidelberg.