The lysosome plays a crucial role in the cellular degradation of biopolymers and in processes, such as apoptosis, autophagy, and cell signaling. Lipophilic small molecules and drugs that carry a basic moiety can accumulate in the lysosome, by passing the lysosomal membrane in their neutral form, getting trapped in the compartment due to protonation at the lower pH (Fig. ). The pathophysiological consequences of this phenomenon termed lysosomotropism are not yet fully understood, but impairment of lysosomal functionality can be linked to phospholipidosis and disturbed cholesterol homeostasis. Figure : Lysosomotropism is primarily believed to be driven by lipophilicity and protonation state of the compound. In this diagram, 'B' is a lysosomotropic compound and 'BH + ' is its protonated state. Illustration inspired by Kuzu et al. (2017). Recently lysosomotropism has especially drawn attention in multiple drug repurposing studies targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This stems from the crucial involvement of cathepsin L, a lysosomal protease, in cleaving the SARS-CoV-2 spike protein and facilitating virus entry into host cells. By the accumulation of lysosomotropic drugs in the lysosome, the compartment's pH rises, rendering proteolytic enzymes inactive and impeding viral replication. Consequently, well-established lysosomotropic drugs, e. g. chloroquine and hydroxychloroquine, were initially promising drug repurposing candidates against SARS-CoV-2. However, these drugs failed to demonstrate significant clinical benefits. In a more general sense, lysosomotropism is also a phenomenon of various drugs. It occurs for structurally different compound classes, modes of actions and targets, and is independent of species and cell-type. For instance, lysosomotropic properties have been observed in anticancer compounds (tamoxifen, doxorubicin, daunorubicin, mitoxantrone), tyrosine kinase inhibitors (imatinib, dasatinib, sunitinib, sorefenib), -blockers (propranolol), antihistamines (promethazine, astemizole, dimebon, desloratadine), and selective serotonin reuptake inhibitors (sertraline, paroxetine, fluoxetine, fluvoxamine). Given such broad range of interests, several groups have contributed towards the measurement, quantification and prediction of lysosomotropism, see e. Highlighting the importance of identifying lysosomotropism early in the development process, Hu et al. (2023) recently developed and published models on phospholipidosis, a process related to lysosomotropism, using compound literature data. They also validated their results using a live-cell imaging assay. Compounds with a calculated logP (clogP) value greater than 2 and a basic pK a (bpKa) value between 6.5 and 11, representing lipophilicity and basicity, respectively, are likely to be lysosomotropic. Here, we will refer to this cross-section of properties as the physicochemical window ("PhysChem window"). While lysosomotropism at first seems to be primarily driven by lipophilicity and protonation state of compounds, it has been established that not all molecules which have the suitable physicochemical properties are in fact lysosomotropic. The Cell Painting Assay (CPA) is an unbiased, image-based phenotypic assay where morphological profiles consisting of hundreds of features are generated from the images of compound-treated and control cells. One typical use case of the CPA is the formation of target hypotheses for test compounds with unknown biological activity. Here profiles of test compounds are compared to those of reference compounds with annotated targets or pathways. However, many compounds with lysosomotropic properties induce a distinct phenotype in the CPA which is independent of their target activity. We have observed that comparing morphological profiles of test compounds to that of a known lysosomotropic agent -Smoothened agonist (SAG), is a reliable surrogate for determining lysosomotropism.