Peripheral blood (PB) immunophenotyping is commonly required for initial evaluation of various suspected disease entities. Several approaches have been proposed. The objective of this work is to explore the value of a 10color protocol developed in our laboratory for flow cytometric assessment of PB leukocytic subsets, as part of a 2tube screening panel. A combination of CD16/CD56/CD34/CD33/CD19/CD4/CD8/CD3/CD20/CD45 antibodies in 1 tube was applied routinely during flow cytometric analysis of PB samples for diagnostic purposes. The protocol was systematically complemented by a 2nd tube with anti-kappa, anti-lambda, CD5, CD19, and CD45 antibodies for adults and selected pediatric patients, and specifically oriented panels when necessary. 25 samples with no detectable neoplastic PB involvement and 31 samples with a hematolymphoid disorder were investigated retrospectively. The contribution of CD33 in the separation of leukocytic populations, as well as the benefits from the simultaneous assessment of CD20/CD19/CD45, CD16/CD56 and the detection of CD34+ cells were examined. The gating strategy with the use of CD33 provided additional information in certain cases. The protocol enabled recognition of differential expression of CD20 and CD45 in CD19+ cells with chronic lymphocytic leukemia phenotype, overall evaluation of NK and NK like T cells, estimation of CD16- granulocytes and CD56/CD16 expression in monocytes, as well as identification of minor cell subsets, such as CD34+ cells. The proposed 10color combination of antibodies analyzed in a standardized manner can offer significant information in the initial evaluation of PB samples, thus, guiding subsequent investigation if needed.