Background/Objectives: Humulus lupulus (hops) are a perennial, dioecious plant widely cultivated for beer production, used for their distinguishing aroma and bitterness—traits that confer high added value status. Various hop-derived compounds have been reported to exhibit antioxidant, antimicrobial, antiproliferative and other bioactive effects. This systematic review and meta-analysis assesses the impact of hop compounds on the viability of diverse cancer cell lines. Methods: A comprehensive literature search was performed following PRISMA guidelines. Data were synthesized via multivariate meta-analysis and meta-regression, using IC₅₀ values as the effect size. Key variables included assay type (SRB, tetrazolium salt-based, crystal violet), exposure duration (24, 48, 72 h), specific hop compound and cancer cell line. Results: Of 622 articles identified, 61 met eligibility criteria, yielding 354 individual experiments. Meta-regression of xanthohumol (XN) IC₅₀ values across SRB, tetrazolium and crystal violet assays revealed no statistically significant differences at 24 h (p = 0.77), 48 h (p = 0.35) and 72 h (p = 0.70), supporting the interchangeability of the methods. Meta-analysis confirmed that hop constituents inhibit cancer cell proliferation; XN emerged as the most potent flavonoid (IC₅₀ = 16.89 μM at 72 h), while lupulone was the strongest compound overall (IC₅₀ = 5.00 μM at 72 h). Crude hop extracts demonstrated greater antiproliferative selectivity for cancer versus non-cancer cells (IC₅₀ = 35.23 vs. 43.80 μg/mL at 72 h). Conclusions: Hop compounds, and particularly bitter acids, demonstrate promising antiproliferative activity against cancer cells with comparatively low toxicity to healthy cells. Furthermore, our analysis confirms the comparability of SRB, tetrazolium-based and crystal violet assays, supporting the robust integration of antiproliferative data.