Selectivity profiling is key for assessing the pharmacological properties of multi-target drugs. We have developed a cell-based and barcoded assay encompassing ten druggable targets, including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), nuclear receptors, a protease as well as their key downstream pathways and profiled 17 drugs in living cells for efficacy, potency, and side effects. Notably, this multiplex assay, termed safetyProfiler assay, enabled the simultaneous assessment of multiple target and pathway activities, shedding light on the polypharmacological profile of compounds. For example, the neuroleptics clozapine, paliperidone, and risperidone potently inhibited primary targets DRD2 and HTR2A as well as cAMP and calcium pathways. However, while paliperidone and risperidone also potently inhibited the secondary target ADRA1A and mitogen-activated protein kinase (MAPK) downstream pathways, clozapine only exhibited mild antagonistic effects on ADRA1A and lacked MAPK inhibition downstream of DRD2 and HTR2A. Furthermore, we present data on the selectivity for bazedoxifene, an estrogen receptor antagonist currently undergoing clinical phase 2 trials for breast cancer, on MAPK signaling. Additionally, precise potency data for LY2452473, an androgen receptor antagonist, that completed a phase 2 clinical trial for prostate cancer, are presented. The non-selective kinase inhibitor staurosporine was observed to potently inactivate the two RTKs EGFR and ERBB4 as well as MAPK signaling, while eliciting stress-related cAMP responses. Our findings underscore the value of comprehensive profiling in elucidating the pharmacological properties of established and novel therapeutics, thereby facilitating the development of novel multi-target drugs with enhanced efficacy and selectivity.