Acrylamide (AA) is a toxin in food systems associated with neurotoxicity, carcinogenicity, genotoxicity, and cardiotoxicity. While several approaches exist, this work details the first-of-its-kind application of Powder X-Ray Diffraction (PXRD) for the detection of AA in food systems. A new methodology was validated as per ICH guidelines. Various x-ray tube combinations such as current (25–45 mA), voltage (25–45 kV), and scanning parameters such as step size (0.006–0.10°) and time-per-step (50–150 s) were experimented on 10–80° angle 2theta (2θ). In this investigation, more diffracted peaks were detected on time per step 151.210 [s], 0.0135[°] step size @ 35 mA/45 kV through phase quantification. The developed method was successfully applied to real processed food samples for qualitative analysis of AA. In addition, the influence of oil frying temperature–time conditions on AA content was studied. Overall, this work highlights the prospects of PXRD-based food quality and safety evaluation.