MOTIVATION: Investigating the molecular composition of different neural compartments such as axons, dendrites, or synapses is critical for understanding learning and memory. State-of-the-art microscopy techniques now resolve individual molecules and pinpoint their position with a micrometer or nanometre resolution across hundreds of micrometres, allowing the labelling of multiple structures of interest simultaneously. Algorithmically, tracking individual molecules across hundreds of micrometres and determining whether they are inside a particular cellular compartment can be challenging. Historically, microscopy images are annotated manually, often using multiple software packages to detect fluorescence puncta and quantify cellular compartments of interest. Advanced ANN-based automated tools, while powerful, often can only help with selected parts of the data analysis, may be optimized for specific spatial resolutions, cell preparations, and may not be fully open source and open access to be sufficiently customizable. RESULTS: Thus, we developed SpyDen, a Python package based upon three principles: (i) ease of use for multi-task scenarios, (ii) open-source accessibility and data export to a standard, open data format, (iii) the ability to edit any software-generated annotation and generalize across spatial resolutions. SpyDen operates on 2D microscopy time-series data, offering robust temporal tracking and spatial analysis capabilities. Equipped with a graphical user interface and accompanied by video tutorials, SpyDen provides a collection of powerful algorithms that can be used for neurite and synapse detection, fluorescent puncta, and intensity analysis. We validated SpyDen using expert annotation across numerous use cases to prove a powerful, integrated platform for efficient and reproducible molecular imaging analysis. AVAILABILITY AND IMPLEMENTATION: SpyDen is available on https://github.com/meggl23/SpyDen while the compiled executables can be found at https://gin.g-node.org/CompNeuroNetworks/SpyDenTrainedNetwork.