The Doppler Shift Spectroscopy (DSS) diagnostic is in the conceptual stage to estimate beam divergence, stripping losses, and beam uniformity of the 100 keV hydrogen Diagnostics Neutral Beam of International Thermonuclear Experimental Reactor. This DSS diagnostic is used to measure the above-mentioned parameters with an error of less than 10%. To aid the design calculations and to establish a methodology for estimation of the beam divergence, DSS measurements were carried out on the existing prototype ion source RF Operated Beam Source in India for Negative ion Research. Emissions of the fast-excited neutrals that are generated from the extracted negative ions were collected in the target tank, and the line broadening of these emissions were used for estimating beam divergence. The observed broadening is a convolution of broadenings due to beam divergence, collection optics, voltage ripple, beam focusing, and instrumental broadening. Hence, for estimating the beam divergence from the observed line broadening, a systematic line profile analysis was performed. To minimize the error in the divergence measurements, a study on error propagation in the beam divergence measurements was carried out and the error was estimated. The measurements of beam divergence were done at a constant RF power of 50 kW and a source pressure of 0.6 Pa by varying the extraction voltage from 4 kV to10 kV and the acceleration voltage from 10 kV to 15 kV. These measurements were then compared with the calorimetric divergence, and the results seemed to agree within 10%. A minimum beam divergence of ∼3° was obtained when the source was operated at an extraction voltage of ∼5 kV and at a ∼10 kV acceleration voltage, i.e., at a total applied voltage of 15 kV. This is in agreement with the values reported in experiments carried out on similar sources elsewhere.