HIV and SARS-CoV-2 are two major viral pathogens of the past 50 years, causing a global HIV/AIDS epidemic in the 1980s and a global COVID-19 pandemic in 2020, respectively. Both the HIV epidemic and COVID-19 pandemic are currently ongoing, and both viruses have infected millions of people with devastating consequences including long-term health effects and death. Like all viruses, HIV and SARS-CoV-2 rely upon host cell machinery to replicate, and characterizing these host-pathogen interactions informs our understanding of virology, pathology, and therapeutics. Host factors identified as critical for viral infection can serve as candidate drug targets for host-directed therapy (HDT), and novel drugs are desperately needed for both HIV and SARS-CoV-2. While anti-retroviral therapy (ART) halts the progression of HIV to AIDS, it is not curative, and ART treatment must be lifelong. While there are effective vaccines against SARS-CoV-2, therapeutics are needed for both unvaccinated and breakthrough infections. HDT, or drugging host factors instead of viral proteins, is an appealing strategy to treat both HIV and SARS-CoV-2 because it limits the potential for viral escape mutations. Systems biology approaches are promising tools to identify drug targets for HDT, as they capture a global and unbiased picture of infection. Here, we employ systems biology approaches to identify host factors important for SARS-CoV-2 and HIV infection that can serve as HDT candidates. In Chapter 2, we propose proteomic approaches to inform SARS-CoV-2 virology, COVID-19 pathology, and therapeutic strategies to combat the current pandemic. Specifically, we discuss how (1) mass spectrometry-based structural techniques can overcome limitations and complement traditional structural approaches to inform the dynamic structure of SARS-CoV-2 proteins, complexes, and virions; (2) virus–host protein–protein interaction mapping can identify the cellular machinery required for SARS-CoV-2 replication; (3) global protein abundance and post-translational modification profiling can characterize signaling pathways that are rewired during infection; and (4) proteomic technologies can aid in biomarker identification, diagnostics, and drug development in order to monitor COVID-19 pathology and investigate treatment strategies. In Chapter 3, we combine proteomic and genetic systems biology approaches to identify E3 ubiquitin ligases critical for HIV infection and latency reversal. We use proteomics to identify 116 E3 ubiquitin ligases that are expressed in physiologically-relevant primary human CD4+ T cells, knock them out with CRISPR-Cas9, and identify 10 E3s that affect HIV infection: TRAF2, TRAF3, and PRPF19 have antiviral activity, and MARCH5, ZFP91, UHRF1, VPS18, NOSIP, PPIL2, RING1 have proviral activity. Importantly, we find that TRAF2 knockouts reverse latency in JLat and primary cell models, thus identifying TRAF2 as a candidate HDT drug target for “shock and kill” therapy towards an HIV cure. Chapter 4 discusses the power of global, unbiased systems biology approaches to elucidate host-pathogen interactions that are critical for understanding the virology of and developing therapeutics for these two major pathogens of the past 50 years.