This study aims to develop an experimental method for determining the focused-sample stream width in microfluidic chips. The focused-sample stream width in the junction of a cross-linked microchannel is determined by the distance between the two streamlines dividing the focusing sheath streams and the sample stream, which should be insensitive to the sample properties. However, the commonly used FWHM intensity method is dependent on the dye properties and cannot be used to estimate the sample focusing effects very accurately. The proposed method is based upon the fact that the cross-stream concentration profile of the sample stream in a region downstream of the junction matches the Gaussian distribution. Therefore, the width of the focused sample stream can be estimated from the channel widths and the variance and maximum concentration of the experimentally measured concentration profile. To validate this method, fluorescein dye has been used to obtain the concentration profile. It has been shown that the proposed method is independent of the fluorescent dye proper-ties. Comparison between the proposed method, FWHM method, and some reported analytical methods also validates the proposed method.