/ AbstractThe Planetary Instrument for X-ray Lithochemistry (PIXL) is a rasterable focused-beam X-ray fluorescence (XRF) spectrometer mounted on the arm of National Aeronautics and Space Administration's (NASA) Mars 2020 Perseverance rover. To ensure that PIXL would be capable of performing accurate in-flight compositional analysis of martian targets, in situ, an elemental calibration was performed pre-flight on the PIXL flight instrument in a simulated martian environment. The details of this calibration, and implications for measuring unknown materials on Mars are the subjects of this paper. The major goals of this calibration were both to align the spectrometer to perform accurate elemental analysis and, to derive a matrix of uncertainties that are applied to XRF measurements of all elements in unknown materials. A small set of pure element and pure compound targets and geologically relevant reference materials were measured with the flight hardware in a simulated martian environment. Elemental calibration and quantifications were carried out using PIXL's XRF quantification software (PIQUANT). Uncertainties generated were implemented into the PIQUANT software version employed by the PIXL's data visualization software (PIXLISE). We outline in this work, a list of factors that impact micro-XRF accuracy, the methodology and steps involved in the calibration, details on the fabrication of the uncertainty matrix, instructions on the use and interpretations of the uncertainties applied to unknowns and an assessment on the limitations and areas open to future improvement as part of subsequent calibration efforts.