•Osprey is an open-source analysis toolbox for magnetic resonance spectroscopy data.•Includes pre-processing, linear-combination modelling, and quantification.•Includes a graphical user interface for visualization of each analysis step. Processing and quantitative analysis of magnetic resonance spectroscopy (MRS) data are far from standardized and require interfacing with third-party software. Here, we present Osprey, a fully integrated open-source data analysis pipeline for MRS data, with seamless integration of pre-processing, linear-combination modelling, quantification, and data visualization. Osprey loads multiple common MRS data formats, performs phased-array coil combination, frequency-and phase-correction of individual transients, signal averaging and Fourier transformation. Linear combination modelling of the processed spectrum is carried out using simulated basis sets and a spline baseline. The MRS voxel is coregistered to an anatomical image, which is segmented for tissue correction and quantification is performed based upon modelling parameters and tissue segmentation. The results of each analysis step are visualized in the Osprey GUI. The analysis pipeline is demonstrated in 12 PRESS, 11 MEGA-PRESS, and 8 HERMES datasets acquired in healthy subjects. Osprey successfully loads, processes, models, and quantifies MRS data acquired with a variety of conventional and spectral editing techniques. Osprey is the first MRS software to combine uniform pre-processing, linear-combination modelling, tissue correction and quantification into a coherent ecosystem. Compared to existing compiled, often closed-source modelling software, Osprey’s open-source code philosophy allows researchers to integrate state-of-the-art data processing and modelling routines, and potentially converge towards standardization of analysis. Osprey combines robust, peer-reviewed data processing methods into a modular workflow that is easily augmented by community developers, allowing the rapid implementation of new methods.