Purpose Primary vitreoretinal lymphoma [(P) VRL ]) is a rare malignancy of the eye localized in the retina, vitreous or choroid. Here, we aim to determine the value of the combination of innovative diagnostic methods for accurate differentiation between (P) VRL and non‐(P) VRL in patients with suspect uveitis or vitritis. Methods Multicolour flow cytometric immunophenotyping of cells in the vitreous samples was performed using the EuroFlow small sample tube. Additionally, cytokines/chemokines and growth factors were measured in the vitreous specimens using a multiplex immunoassay. Data were evaluated in predefined clinical subgroups using omniviz unsupervised Pearson's correlation visualization and unsupervised heatmap analysis. Results A total of 53 patients were prospectively included in the period 2012–2015. In the (P) VRL subgroup ( n = 10), nine cases showed aberrant surface membrane immunoglobulin (SmIg) light chain expression. In the non‐(P) VRL group ( n = 43) clearly skewed SmIg light chain expression was observed in two multiple sclerosis‐related uveitis cases, but not in other uveitis types. Soluble mediator measurement revealed high interleukin ( IL )‐10/ IL ‐6 ratios, and high IL ‐1 RA levels in 9/10 (P) VRL cases, but not in any non‐(P) VRL case. Further correlation and heatmap analysis revealed a minimal signature of cellular parameters ( CD 19+ B cells, aberrant SmIg light chain expression) and cytokine parameters ( IL ‐10/ IL ‐6 ratio >1, high IL ‐10, high IL ‐1 RA , high monocyte chemotactic protein‐1, high macrophage inflammatory protein‐1 β ) to reliably distinguish (P) VRL from non‐(P) VRL . Conclusion Here, we show the power of a combined cellular and proteomics strategy for detecting (P) VRL in vitreous specimens, especially in cases with minor cellular (P) VRL infiltrates.